Inhibitors of rho associated coiled-coil containing protein kinase

ABSTRACT

The invention relates to inhibitors of ROCK1 and/or ROCK2. Also provided are methods of inhibiting ROCK1 and/or ROCK2 that are useful for the treatment of disease.

FIELD OF THE INVENTION

The invention relates to inhibitors of ROCK1 and/or ROCK2. Also providedare methods of inhibiting ROCK1 and/or ROCK2 that are useful for thetreatment of disease.

BACKGROUND OF THE INVENTION

Rho Associated Coiled-Coil Containing Protein Kinases (ROCK) are membersof the serine/threonine kinase family. Two isoforms, ROCK 1 and ROCK 2,have been identified. Both isoforms are activated by GTP-bound forms ofRho GTPase and when activated phosphorylate a variety of downstreamsubstrates. ROCKs play important roles in numerous cellular processesincluding smooth muscle cell contraction, cell proliferation, adhesionand migration. As such, ROCK inhibitors have potential therapeuticapplicability in a wide variety of pathological conditions including,for example, asthma, cancer, erectile dysfunction, glaucoma, insulinresistance, kidney failure, pulmonary hypertension, neuronaldegeneration, and osteoporosis.

ROCK is a key intracellular regulator of cytoskeletal dynamics and cellmotility. ROCK regulates a number of downstream targets of RhoA throughphosphorylation, including, for example, myosin light chain, the myosinlight chain phosphatase binding subunit and LIM-kinase 2. Thesesubstrates regulate actin filament organization and contractility. Insmooth muscle cells, ROCK mediates calcium sensitization and smoothmuscle contraction. Inhibition of Rho-kinase blocks 5-HT andphenylephrine agonist induced muscle contraction. When introduced intonon-smooth muscle cells, ROCK induces stress fiber formation and isrequired for the cellular transformation mediated by RhoA. ROCKparticipates in a variety of cellular processes, including but notlimited to cell adhesion, cell motility and migration, growth control,cell contraction, and cytokinesis. ROCK is also involved in Na/Hexchange transport system activation, stress fiber formation, adducinactivation, and physiological processes such as vasoconstriction,bronchial smooth muscle constriction, vascular smooth muscle andendothelial cell proliferation, platelet aggregation, and others.

Inhibition of ROCK activity in animal models has demonstrated a numberof benefits of ROCK inhibition for the treatment of human diseases.These include models of cardiovascular diseases such as hypertension,atherosclerosis, restenosis, cardiac hypertrophy, ocular hypertension,cerebral ischemia, cerebral vasospasm, penile erectile dysfunction,central nervous system disorders such as neuronal degeneration andspinal cord injury, and in neoplasias. Inhibition of ROCK activity hasbeen shown to inhibit tumor cell growth and metastasis, angiogenesis,arterial thrombotic disorders such as platelet aggregation and leukocyteaggregation, asthma, regulation of intraocular pressure, and boneresorption. The inhibition of ROCK activity in patients has benefits forcontrolling cerebral vasospasms and ischemia following subarachnoidhemorrhage, reduction of intraocular pressure, increase in ocularaqueous outflow by relaxation of trabecular meshwork tissue, improvingblood flow to the optic nerve, and protection of healthy ganglion cells.

A substantial body of in vivo data has been generated focusing on theactivity of ROCK in the CNS. Abnormal activation of the ROCK pathway hasbeen documented in many disorders of the central nervous system. Forexample, axon growth and synaptic plasticity are dependent on thestructural regulation of the actin cytoskeleton. The Rho-ROCK cascadehas a central role in synaptic plasticity, both in dendritemorphogenesis and stability as well as in growth cone motility andcollapse. In addition, multiple axon growth inhibitory moleculesconverge on RhoA/ROCK in neurons making this an attractive pathway forintervention in CNS disorders.

Nogo-receptors (NgR) (along with other complex members, includingLINGO-1) and their ligands are perhaps the most well characterized andpotent inhibitors of neurite outgrowth. Some of the earliest eventsdownstream of receptor activation by the myelin-associated inhibitorsare the upregulation of RhoA and ROCKs. These events lead to increasedcontractility and have strong inhibitory effects on axonal growth inmature neurons. Thus, the possibility of inhibiting this signal cascadeprovides a very promising therapeutic strategy in Spinal Cord andOptical Nerve injuries. Neurodegenerative conditions such asHuntington's and Alzheimer's (AD) disease are also being investigated asresponsive to inhibition of the NgR signaling. Not only do NgR familymembers associate with APP processing but also subcellular localizationof NgR and Nogo is altered in AD brain.

Alzheimer's disease (AD), the most common cause of dementia in theelderly, is a progressive neurodegenerative disorder, which involves agradual decline of many cognitive functions including memory impairment(Selkoe, 2001). Synaptic loss is generally observed in AD pathology andis the hallmark of synaptic dysfunction in AD (Tanzi and Bertram, 2005).Oligomerized β-amyloid peptides have been implicated in the loss ofsynaptic plasticity and neural network dysfunction. Synaptic plasticityis dependent on the structural regulation of the actin cytoskeleton indendritic spines. Rho-ROCK cascade has a central role in synapticplasticity, both in dendrite morphogenesis and stability as well as ingrowth cone motility and collapse (Govek et al., 2005; Linseman andLoucks, 2008). Several studies have demonstrated that ROCK kinases caninduce generation of toxic β-amyloid peptide and furthermore, inhibitionof ROCKs, can inhibit toxic peptide processing. In a feed-forwardmechanism, β-amyloid increases the Rho GTPase activity, which via ROCKactivation inhibits neurite outgrowth and synapse formation (Petratos etal., 2008). Thus, ROCK inhibitors may hold the potential for preventingsynaptic and neuronal degradation as well as for promoting regenerativeprocesses in AD. A recent study by Herskowitz et al. showed that ROCKknockdown decreased αβ levels. These effects demonstrate that highlyROCK selective inhibitors are needed to provide an effective treatmentof Alzheimer's disease (AD). A model compound SR3677 was tested in arodent model of AD, in an effort to demonstrate the use of ROCKinhibition for AD, by altering BACE-1 distribution and amyloid precursorprotein (APP) trafficking to lysosomes. After a direct i.p. injectioninto hippocampus due to its poor oral PK properties (5% F and <1 hrhalf-life) and lack of brain penetration, SR3677 had the promisingeffect of lowering sAPPP.

Huntington's disease (HD) is a devastating, untreatable, dominantlyinherited neurodegenerative disease characterized by psychiatricdisturbance, motor impairment, and dementia. Misfolding and aggregationof the Htt protein, a product of the huntingtin gene, causes the HDpathology (Shao and Diamond, 2007). Very few mechanism-based therapeuticleads for treatment of HD have been developed. While scientificinvestigations are still ongoing, multiple lines of evidence suggestthat ROCK inhibition may constitute an effective treatment for HD. Inmouse models of HD, ROCK inhibition significantly reduced soluble Httlevels, reversed aggregate formation, neurite retraction and wasprotective against neuronal cell death (Deyts et al., 2009; Li et al.,2009). Similar results were obtained in Drosophila studies, whereinhibition of ROCK controlled Htt aggregation (Shao et al., 2008a; Shaoet al., 2008b). The ROCK signaling pathway is a promising therapeutictarget for HD.

ROCK signaling has also been implicated in Parkinson's disease andamyotrophic lateral sclerosis (ALD). See, e.g., Tonges, L. et al.(2012). “Inhibition of rho kinase enhances survival of dopaminergicneurons and attenuates axonal loss in a mouse model of Parkinson'sdisease.” Brain. 135(11):3355-70.

ROCKs phosphorylate multiple downstream substrates, includingmyosin-light-chain (MLC, at threonine 18 and serine 19) and myosinlight-chain phosphatase (MYPT1, at threonine 853), to drive thepolymerization of globular G-actin into filamentous F-actin and assembleactomyosin contractile machinery. It has been recognized that thispathway is may contribute to the pathogenesis of several CNS disorderssuch as spinal cord injuries, stroke, and AD. In the adult CNS, injuredaxons regenerate poorly due to the presence of myelin-associated axonalgrowth inhibitors. Myelin-associated inhibitors such asmyelin-associated glycoprotein (MAG), Nogo, oligodendrocyte-myelinglycoprotein (OMgp) and repulsive guidance molecule (RGM) limit axonalregeneration in the injured brain and spinal cord. A common mechanismfor various myelin-associated inhibitors is that they all activate Rhoand its downstream effector kinase ROCKs to inhibit neurite outgrowth.

Blockade of Rho/ROCK pathway by small molecule is a desirable strategyin central nervous system (CNS) disorders. However, the blood-brainbarrier (BBB) while serving a critical role in brain homeostasis, alsosignificantly impedes the penetration of most small molecule inhibitors.With growing interest in developing selective and potent inhibitors forthe treatment of CNS diseases, there is an urgent need for inhibitors ofROCK 1 and/or ROCK 2, in particular those that cross the blood-brainbarrier.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of formula I

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   R² is selected from the group consisting of aryl, heteroaryl,    aralkyl, and heterocyclyl, each of which may be unsubstituted or    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro    alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RO₂C—, aryl-O—    and heteroaryl-O—;-   alternatively, R¹ and R² taken together form a monocyclic group or a    bicyclic group, wherein the monocyclic group has 4 to 7 ring atoms,    including up to 2 ring heteroatoms, and the bicyclic group has 8 to    10 ring atoms, including up to 3 ring heteroatoms, and wherein the    monocyclic group and bicyclic group are unsubstituted or are    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, aryl and heteroaryl;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   R⁵ is selected from H, lower alkyl and C₃-C₆ cycloalkyl;-   a is 0 or 1;-   b is 0 to 2;-   and-   each R and R′ is independently selected from H, lower alkyl, and    C₃-C₆ cycloalkyl, or alternatively, R and R′ taken together form a 5    to 6 membered heterocyclic ring.

The present invention includes pharmaceutical compositions comprisingthe compounds of the invention and a pharmaceutically acceptablecarrier.

The present invention includes compositions comprising a substantiallypure compound of the invention and a pharmaceutically acceptable salt,stereoisomer, or hydrate thereof, and a pharmaceutically acceptablecarrier.

In one aspect, the invention provides a method of inhibiting a ROCK in amammal comprising administering an effective amount of one or morecompounds of Formula I. The invention provides a method of treating apatient suffering from a disease comprising administering to the patientin need of such treatment a therapeutically effective amount of acompound of Formula I. In certain such embodiments, the compound ofFormula I inhibits ROCK1 and/or ROCK2. In certain such embodiments, thecompound of Formula I selectively inhibits ROCK1 and/or ROCK2.Non-limiting diseases and conditions treated according to the instantinvention include central nervous system disorders such as neuronaldegeneration and spinal cord injury, cardiovascular diseases such ashypertension, atherosclerosis, restenosis, cardiac hypertrophy, ocularhypertension, cerebral ischemia, cerebral vasospasm, penile erectiledysfunction, arterial thrombotic disorders such as platelet aggregationand leukocyte aggregation, asthma, regulation of intraocular pressure,and bone resorption. In neoplasias, inhibition of Rho-kinase inhibitstumor cell growth and metastasis, and angiogenesis.

The invention provides a method of treating a central nervous systemdisorder in a subject comprising administering to the subject atherapeutically effective amount of a compound of Formula I. Centralnervous system disorders include, without limitation, neuronaldegeneration or spinal cord injury, as well as Huntington's disease,Parkinson's disease, Alzheimer's disease, Amyotrophic lateral sclerosis(ALS), or multiple sclerosis.

The invention provides a method of treating an autoimmune disorder in asubject comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula I. Autoimmune disordersinclude, without limitation, rheumatoid arthritis, (multiple sclerosis),systemic lupus erythematosus (SLE; lupus), psoriasis, Crohn's disease,atopic dermatitis, eczema, or graft-versus-host disease (GVHD).

The invention provides a method of treating a cardiovascular disorder ina subject comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula I. Cardiovascular disordersinclude, without limitation, hypertension, atherosclerosis, angina,arterial obstruction, peripheral arterial disease, peripheralcirculatory disorder, cerebral cavernous malformation, restenosis,cardiac hypertrophy, ocular hypertension, cerebral ischemia, cerebralvasospasm, acute respiratory distress syndrome (ARDS) or erectiledysfunction.

The invention provides a method of treating inflammation in a subjectcomprising administering to the subject a therapeutically effectiveamount of a compound of Formula I. Inflammation includes, withoutlimitation, asthma, cardiovascular inflammation, renal inflammation orarteriosclerosis.

The invention provides a method of treating an arterial thromboticdisorder in a subject comprising administering to the subject atherapeutically effective amount of a compound of Formula I.Non-limiting examples of arterial thrombotic disorders are plateletaggregation, or leukocyte aggregation.

The invention provides a method of treating a fibrotic disorder in asubject comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula I. Non-limiting examples offibrotic disorders are pulmonary fibrosis including cystic andidiopathic pulmonary fibrosis, radiation induced lung injury, liverfibrosis including cirrhosis, cardiac fibrosis including arterialfibrosis, endomyocardial fibrosis, old myocardial infraction, arterialstiffness, atherosclerosis, restenosis, arthrofibrosis, Crohn's disease,myelofibrosis, Peyronie's diseases, nephrogenic systemic fibrosis,progressive massive fibrosis, retroperitoneal cavity fibrosis,schleroderma/systemic sclerosis, mediastinal fibrosis, Keloids andhypertrophic scars, glial scaring, or renal fibrosis.

The invention provides a method of maintaining epithelial stabilitycomprising administering to the subject a therapeutically effectiveamount of a compound of Formula I.

The invention provides a method of treating glaucoma or regulatingintraocular pressure in a subject comprising administering to thesubject a therapeutically effective amount of a compound of Formula I.Non-limiting examples of glaucoma include primary open-angle glaucoma,acute angle-closure glaucoma, pigmentary glaucoma, neovascular glaucoma,congenital glaucoma, normal tension glaucoma, or secondary glaucoma.

The invention provides a method of treating a neoplastic disease in asubject comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula I. Neoplastic diseasesinclude, without limitation, a lymphoma, carcinoma, leukemia, sarcoma,or blastoma, such as squamous cell cancer, small-cell lung cancer,pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma,non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, braincancer, endometrial cancer, testis cancer, cholangiocarcinoma,gallbladder carcinoma, gastric cancer, melanoma, or head and neckcancer.

The invention also provides a method of treating metabolic syndrome,insulin resistance, hyperinsulinemia, type 2 diabetes, or glucoseintolerance in a subject comprising administering to the subject atherapeutically effective amount of a compound of Formula I.

Further, the invention provides a method of treating osteoporosis orpromoting bone formation a subject comprising administering to thesubject a therapeutically effective amount of a compound of Formula I.

The invention provides a method of treating an ocular disorder having anangiogenic component comprising administering to the subject atherapeutically effective amount of a compound of Formula I and anangiogenesis inhibitor. Non-limiting examples of such ocular disordersinclude age related macular degeneration (AMD), choroidalneovascularization (CNV), diabetic macular edema (DME), irisneovascularization, uveitis, neovascular glaucoma, or retinitis ofprematurity (ROP).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Representative Z′-Lyte assay results. The ROCK inhibitors of thepresent invention show as low as single digit nanomolar potenciestowards both isoforms of ROCK.

FIG. 2. Compounds of the present invention inhibit both ROCK isoforms incells. HCT116 cells with either a ROCK1 or ROCK2 knockout (ROCK1KO andROCK2KO, respectively) were treated with the inhibitor for 90 min andpMypt (T853) levels were visualized by western blotting. The ROCKinhibitor efficiently blocked ROCK target MYPT1 phosphorylation at 110nM.

FIG. 3. Human oligodendrocyte/neuron progenitor cells were cultured invitro with or without ROCK inhibitor (Example 2 compound) for 2 & 14days. Nestin and MAP2 proteins were visualized by staining withcommercial antibodies to identify different stages of neuronal celldifferentiation. The ROCK inhibitor significantly facilitated matureneuronal cell marker MAP2 expression while improved neurite outgrowth,as evidenced by strong increase in MAP2 signal in cells differentiatedin the presence of the ROCK inhibitor.

FIG. 4. Under co-cultured condition of rat oligodendrocytes with ratdorsal root ganglia (DRG) explants, treatment with the ROCK inhibitor(Example 2 compound) changed cytoskeletal organization, producing manyshorter ordered myelin segments, which were identified by neurofilamentstaining. At the same time, the ROCK inhibitor also facilitated axonalsupport of oligodendrocyte, which was demonstrated by the alignment ofoligodendrocyte (stained for MBP) along with the direction of axonal(stained for neurofilament) extension.

DETAILED DESCRIPTION OF THE INVENTION

ROCK Inhibitors

Compounds according to the present invention include those having theformula I:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   R² is selected from the group consisting of aryl, heteroaryl,    aralkyl, and heterocyclyl, each of which may be unsubstituted or    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro    alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RO₂C—, aryl-O—    and heteroaryl-O—;-   alternatively, R¹ and R² taken together form a monocyclic group or a    bicyclic group, wherein the monocyclic group has 4 to 7 ring atoms,    including up to 2 ring heteroatoms, and the bicyclic group has 8 to    10 ring atoms, including up to 3 ring heteroatoms, and wherein the    monocyclic group and bicyclic group are unsubstituted or are    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, aryl and heteroaryl;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   R⁵ is selected from H, lower alkyl and C₃-C₆ cycloalkyl;-   a is 0 or 1;-   b is 0 to 2;-   and-   each R and R′ is independently selected from H, lower alkyl, and    C₃-C₆ cycloalkyl, or alternatively, R and R′ taken together form a 5    to 6 membered heterocyclic ring.

Compounds according to the present invention include those having theformula II:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   R² is selected from the group consisting of aryl, heteroaryl,    aralkyl, and heterocyclyl, each of which may be unsubstituted or    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro    alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RO₂C—, aryl-O—    and heteroaryl-O—;-   alternatively, R¹ and R² taken together form a monocyclic group or a    bicyclic group, wherein the monocyclic group has 4 to 7 ring atoms,    including up to 2 ring heteroatoms, and the bicyclic group has 8 to    10 ring atoms, including up to 3 ring heteroatoms, and wherein the    monocyclic group and bicyclic group are unsubstituted or are    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, aryl and heteroaryl;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   b is 0 to 2; and-   each R and R′ is independently selected from H, lower alkyl, and    C₃-C₆ cycloalkyl, or alternatively, R and R′ taken together form a 5    to 6 membered heterocyclic ring.

In a certain embodiments of the present invention, there is provided acompound of the formula III:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   R² is selected from the group consisting of aryl, heteroaryl,    aralkyl, and heterocyclyl, each of which may be unsubstituted or    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro    alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RO₂C—, aryl-O—    and heteroaryl-O—;-   alternatively, R¹ and R² taken together form a monocyclic group or a    bicyclic group, wherein the monocyclic group has 4 to 7 ring atoms,    including up to 2 ring heteroatoms, and the bicyclic group has 8 to    10 ring atoms, including up to 3 ring heteroatoms, and wherein the    monocyclic group and bicyclic group are unsubstituted or are    optionally substituted with 1 to 3 substituents independently    selected from the group consisting of halo, hydroxy, lower alkyl,    lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, aryl and heteroaryl;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-; and-   each R and R′ is independently selected from H, lower alkyl, and    C₃-C₆ cycloalkyl, or alternatively, R and R′ taken together form a 5    to 6 membered heterocyclic ring.

In a certain embodiments of the present invention, there is provided acompound of the formula IV:

wherein:

-   ring A is a 5- or 6-membered aromatic ring which optionally contains    up to 3 ring heteroatoms;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   b is 0 to 2;-   R⁶ is selected from the group consisting of H, halo, lower alkyl,    substituted lower alkyl, lower alkoxy, amino, hydroxyl, and    carboxyl;-   R⁷ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, and RCONR′—;-   each R and R′ is independently selected from H, lower alkyl, and    C3-C6 cycloalkyl, or alternatively, R and R′ taken together form a 5    to 6 membered heterocyclic ring; and m is 1 to 3.

In a certain embodiments of the present invention, there is provided acompound of the formula V:

wherein:

-   ring B is a 5- or 6-membered aromatic ring which optionally contains    up to 3 ring heteroatoms;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   b is 0 to 2;-   R⁸ is selected from the group consisting of H, halo, lower alkyl,    substituted lower alkyl, lower alkoxy, amino, hydroxyl and carboxyl;-   R⁹ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy and carboxyl, RR′N—, RR′NCO—, RCONH—, and RCONR′—;-   each R and R′ is independently selected from H, lower alkyl, and    C3-C6 cycloalkyl, or alternatively, R and R′ taken together form a 5    to 6 membered heterocyclic ring; and m is 1 to 3.

In a certain embodiments of the present invention, there is provided acompound of the formula VI:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   R³ is selected from H, lower alkyl, substituted lower alkyl, and    RR′N—(C₂₋₄ alkyl)-;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   b is 0 to 2;-   each R²¹ is independently selected from the group consisting of H,    halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃    perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—,    RCONH—, RCONR′—, RO₂C—, aryl-O— and heteroaryl-O—; and-   n is 0 to 3.

In a certain embodiments of the present invention, there is provided acompound of the formula VII:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   R⁴ is selected from the group consisting of H, halo, hydroxy, lower    alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃    perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,    RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;-   b is 0 to 2;-   each R²¹ is independently selected from the group consisting of H,    halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃    perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—,    RCONH—, RCONR′—, RO₂C—, aryl-O— and heteroaryl-O—; and-   n is 0 to 3.

In a certain embodiments of the present invention, there is provided acompound of the formula VIII:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   each R²¹ is independently selected from the group consisting of H,    halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃    perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—,    RCONH—, RCONR′—, RO₂C—, aryl-O— and heteroaryl-O—; and-   n is 0 to 3.

In a certain embodiments of the present invention, there is provided acompound of the formula IX:

wherein:

-   R¹ is selected from the group consisting of lower alkyl, substituted    lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,    R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— and    R¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—;    -   each R¹⁰ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹¹ is independently selected from H, lower alkyl, and        C₃-C₆ cycloalkyl;    -   each R¹² is independently selected from H and lower alkyl;    -   each R¹³ is independently selected from H and lower alkyl;    -   additionally or alternatively, an R¹² and an R¹³ attached to the        same carbon atom may be taken together to form a C₃-C₆        cycloalkyl group;    -   W is a 3- to 7-membered heterocyclic ring having 1 to 3 ring        heteroatoms;    -   c is 2 to 4;    -   d is 1 to 4;-   each R²² is independently selected from the group consisting of H,    halo, hydroxy, lower alkyl, lower alkoxy, amino, C₁-C₃ perfluoro    alkyl, and C₁-C₃ perfluoro alkoxy; and-   n is 0 to 3.

In preferred embodiments for the Formulas I to IX, R¹ is selected to belower alkyl. More preferably, R¹ is C₁ to C₃ alkyl, and still morepreferably, R¹ is methyl or ethyl.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 8 or fewer carbon atoms inits backbone (e.g., C1-C8 for straight chain, C3-C8 for branched chain),and more preferably 6 or fewer. Likewise, preferred cycloalkyls havefrom 3-8 carbon atoms in their ring structure, and more preferably have3 to 6 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto four carbons, and more preferably from one to three carbon atoms. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl. Lower alkyl groups include methyl, ethyl, propyl,isopropyl, n-butyl, iso-butyl, and tert-butyl.

The term “cycloalkyl” refers to saturated, carbocyclic groups havingfrom 3 to 6 carbons in the ring. Cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl.

The term “substituted alkyl” refers to an alkyl group as defined above,and having one to three substituents. The substituents are selected fromthe group consisting of halo, hydroxy, lower alkoxy, amino, lower alkylamino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy andcarboxyl.

“Substituted lower alkyl” refers to a lower alkyl group as definedabove, and having one to three substituents. The substituents areselected from the group consisting of halo, hydroxy, lower alkoxy,amino, nitro, cyano, perfluoro lower alkyl, perfluoro lower alkoxy andcarboxyl.

“Substituted cycloalkyl,” such as “substituted C₃-C₆ cycloalkyl” refersto a cycloalkyl group as defined above, and having one to threesubstituents. The substituents are selected from the group consisting ofhalo, hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, perfluorolower alkyl, perfluoro lower alkoxy and carboxyl.

As used herein, the term “halogen” or “halo” designates —F, —Cl, —Br or—I, and preferably F, Cl or Br.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, that is attached through an oxygen atom.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. The term “lower alkoxy” refers to an alkoxysubstituent in which a lower alkyl is bonded through an oxygen atom,wherein the “lower alkyl” portion is as defined above.

The terms “amine” and “amino” refer to both unsubstituted andsubstituted amines, e.g., a moiety that can be represented by thegeneral formula:

wherein R and R′ are each independently selected from H and lower alkyl.

The term “aryl” as used herein includes 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like. Those aryl groups having heteroatoms in thering structure may also be referred to as “aryl heterocycles” or“heteroaryl” groups. The aromatic ring can be substituted at one or morering positions with such substituents as described above. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group. Preferably, the alkyl group is a lower alkyl, asdescribed above.

The term “heterocycle” of “heterocyclyl” refer to non-aromaticheterocycles having from 4 to 7 ring atoms and including from 1 to 3ring heteroatoms.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur. Most preferred are nitrogen and oxygen.

As used herein, the definition of each expression, e.g. alkyl, m, n, R¹,R², etc., when it occurs more than once in any structure, is intended tobe independent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M.Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including c is- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are included in this invention. The inventionalso contemplates the substitution of isotopes of the atoms for thecompounds, for example deuterium for hydrogen, etc.

In one aspect, the present invention provides compounds of Formulas I-IXthat are inhibitors of ROCK. ROCK is found in two forms, ROCK 1 (ROCKβ;p160-ROCK) and ROCK 2 (ROCKα). In some embodiments, the compound ofFormulas I-IX selectively inhibits ROCK1. In some embodiments, thecompound of Formulas I-IX selectively inhibits ROCK2. In someembodiments, the compound of Formulas I-IX is non-selective with respectto inhibition of ROCK1 and ROCK2. In the context of this invention,selective means the inhibitor demonstrates an IC₅₀ that is at least2-fold, at least 5-fold, at least 10-fold, or at least 25-fold lower forone kinase as compared to the IC₅₀ for the other kinase.

Methods of determining kinase inhibition are known in the art. Forexample, kinase activity of an enzyme and the inhibitory capacity of atest compound can be determined by measuring enzyme specificphosphorylation of a substrate. Commercial assays and kits are availableand can be employed. For example, kinase inhibition can be determinedusing an IMAP® assay (Molecular Devices). This assay method involves theuse of a fluorescently tagged peptide substrate. Phosphorylation of thetagged peptide by a kinase of interest promotes binding of the peptideto a trivalent metal-based nanoparticle via the specific, high affinityinteraction between the phospho-group and the trivalent metal. Proximityto the nanoparticle results in increased fluorescence polarization.Inhibition of the kinase by a kinase inhibitor prevents phosphorylationof the substrate and thereby limits binding of the fluorescently-taggedsubstrate to the nanoparticle. Such an assay can be compatible with amicrowell assay format, allowing simultaneous determination of IC₅₀ ofmultiple compounds.

Methods of Treating Disease

In one aspect of the present invention there is provided a method oftreating a patient suffering from a disease comprising administering toa patient in need of such treatment a therapeutically effective amountof a compound of the present invention. The phrase“therapeutically-effective amount” as used herein means that amount of acompound, material, or composition comprising a compound of the presentinvention which is effective for producing some desired therapeuticeffect in at least a sub-population of cells in an animal at areasonable benefit/risk ratio applicable to any medical treatment, e.g.reasonable side effects applicable to any medical treatment.

CNS Disorders

Compounds of Formulas I-IX demonstrate effective blood brain barrier(BBB) penetration, and distribution to tissues of the central nervoussystem. Thus, the compounds of the invention are useful for treatment ofcentral nervous system disorders, as well as disorders, such as certainocular disorders, that benefit from the ability to cross the BBB. Suchdisorders may involve neuronal degeneration or physical injury to neuraltissue, including without limitation, Huntington's disease, Parkinson'sDisease, Alzheimer's, Amyotrophic lateral sclerosis (ALS), Battendisease, dementia, spinal muscular atrophy, motor neurone diseases,spinocerebellar ataxia, acute or chronic pain, dementia, neuronaldegeneration, spinal cord injury, cerebral vasospasm or multiplesclerosis.

Cardiovascular and Other Diseases

Compounds of the invention that inhibit ROCK and/or ROCK mediatedphosphorylation are useful for treatment of patients suffering fromcardiovascular and non-cardiovascular diseases involving Rho-kinasefunction, such as hypertension, pulmonary hypertension, atherosclerosis,restenosis, coronary heart disease, cardiac hypertrophy, ocularhypertension, retinopathy, ischemic diseases, cerebral ischemia,cerebral vasospasm, penile erectile dysfunction, peripheral circulatorydisorder, peripheral artery occlusive disease, glaucoma, (e.g.,regulating intraocular pressure), fibroid lung, fibroid liver, fibroidkidney, chronic obstructive pulmonary disease (COPD), adult respiratorydistress syndrome, central nervous system disorders such as neuronaldegeneration and spinal cord injury. Further, ROCK inhibiters of theinvention can be used to treat arterial thrombotic disorders such asplatelet aggregation and leukocyte aggregation, and bone resorption.

In an embodiment of the invention, compounds are used to treat cerebralcavernous malformation (CCM). CCMs are vascular lesions consisting ofclusters of leaky, dilated capillaries and are associated with centralnervous system (CNS) disorders, including seizures and stroke. The lossof vascular integrity is thought to involve activation of RhoA andactivation of ROCK, leading to changes in cytoskeletal stability andincreased vascular permeability. The compounds of the invention inhibitROCK activation and restore vascular endothelialfunction.

Glaucoma

In an embodiment of the invention, a compound of Formulas I-IX is usedto treat glaucoma. The two most common, primary open-angle glaucoma andacute angle-closure glaucoma, are characterized by high ocular pressure.Pigmentary glaucoma and congenital glaucoma also are characterized byreduced fluid outflow and high intraocular pressure (IOP). Normaltension glaucoma is thought to be due to another mechanism, inparticular poor blood flow to the optic nerve. Secondary glaucoma canresult from injury, infection, inflammation, tumor or cataracts, and isalso associated with prolonged use of steroids, systemic hypertension,diabetic retinopathy, and central retinal vein occlusion. Glaucomashaving a neovascular component can benefit from administration of anangiogenesis inhibitor in addition to a ROCK inhibitor.

Inflammation

The invention provides a method of treating inflammation in a subjectcomprising administering to the subject a therapeutically effectiveamount of a compound of Formulas I-IX. Inflammation includes, withoutlimitation, asthma, cardiovascular inflammation, renal inflammation,atherosclerosis and arteriosclerosis, and sepsis. Other inflammatoryconditions that can be treated by methods of the invention includefibrotic conditions (including, e.g., idiopathic pulmonary fibrosis,NASH, scleroderma, systemic sclerosis, and cirrhosis).

Autoimmune Disorders

The invention provides a method of treating an autoimmune disorder in asubject comprising administering to the subject a therapeuticallyeffective amount of a compound of Formulas I-IX. Autoimmune disordersinclude, without limitation, rheumatoid arthritis, multiple sclerosis,systemic lupus erythematosus (SLE; lupus), psoriasis, Crohn's disease,atopic dermatitis, eczema, or graft-versus-host disease (GVHD), AcuteDisseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, Agammaglobulinemia, Alopeciaareata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema,Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmunehepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency,Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmuneoophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmunethrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmuneurticaria, Axonal & neuronal neuropathies, Balo disease, Behcet'sdisease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiacdisease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatorydemyelinating polyneuropathy (CIDP), Chronic recurrent multifocalostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricialpemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome,Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis,CREST disease, Essential mixed cryoglobulinemia, Demyelinatingneuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease(neuromyelitis optica), Discoid lupus, Dressler's syndrome,Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis,Erythema nodosum, Experimental allergic encephalomyelitis, Evanssyndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis(temporal arteritis), Giant cell myocarditis, Glomerulonephritis,Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerlycalled Wegener's Granulomatosis), Graves' disease, Guillain-Barresyndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolyticanemia, Henoch-Schonlein purpura, Herpes gestationis,Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgAnephropathy, IgG4-related sclerosing disease, Immunoregulatorylipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenilearthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis,Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis,Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgAdisease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease,Microscopic polyangiitis, Mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myastheniagravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's),Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromicrheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric DisordersAssociated with Streptococcus), Paraneoplastic cerebellar degeneration,Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis),Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis,Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, &III autoimmune polyglandular syndromes, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primarysclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathicpulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia,Raynaud's phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy,Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome,Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis,Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren'ssyndrome, Sperm & testicular autoimmunity, Stiff person syndrome,Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympatheticophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cellarteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome,Transverse myelitis, Type 1 diabetes, Ulcerative colitis,Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis,Vesiculobullous dermatosis, and Vitiligo.

According to the invention, targeting Th17 (IL-17-secreting) cells byROCK inhibition provides a method for treating Th17 cell-mediateddiseases, including but not limited to autoimmune disorders such as RA,MS, SLE, Psoriasis, and Crohn's disease, and GVHD in humans. In anembodiment of the invention, the ROCK inhibitor is a compound of FormulaI.

The development and function of Tregs depend on activation of specificsignaling transduction pathways. TGF-β and TL-2 activate expression ofFoxp3 and STAT5 transcription factors that both play an essential rolein the control of Treg suppressive function. On the other hand,pro-inflammatory cytokines inhibit Foxp3 expression via up-regulation ofSTAT3 phosphorylation. According to the invention, pharmacologicalinhibition of ROCK2 may regulate Treg function.

Neoplastic Disease

ROCK inhibitors of the invention inhibit tumor cell growth andmetastasis, and angiogenesis, and are useful for treating neoplasticdiseases. Neoplastic diseases include any malignant growth or tumorcaused by abnormal or uncontrolled cell division, and may spread toother parts of the body through the lymphatic system or the bloodstream. Neoplastic disease includes, without limitation, lymphoma (aneoplasm of lymph tissue that is usually malignant), carcinoma (anymalignant tumor derived from epithelial tissue), leukemia (malignantneoplasm of blood-forming tissues; characterized by abnormalproliferation of leukocytes), sarcoma (a usually malignant tumor arisingfrom connective tissue (bone or muscle etc.), and blastoma (malignancyin precursor cells). Nonlimiting examples include squamous cell cancer,small-cell lung cancer, pituitary cancer, esophageal cancer,astrocytoma, soft tissue sarcoma, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer,testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastriccancer, melanoma, and various types of head and neck cancer.

Weight Gain/Loss

According to the invention, ROCK inhibitors are used to effect weightloss and/or limit weight gain. ROCK inhibitors promote weight loss innormal subjects, and limit weight gain in subjects prone to obesity.

Insulin Resistance

In an embodiment of the invention, a ROCK inhibitor is used to reduce orprevent insulin resistance or restore insulin sensitivity. Accordingly,in one embodiment, the compounds of the invention are used to promote orrestore insulin-dependent glucose uptake. In another embodiment of theinvention, a ROCK-inhibitors of the invention is used to promote orrestore glucose tolerance. In another embodiment of the invention, aROCK inhibitor of the invention is used to treat metabolic syndrome. Inanother embodiment, ROCK inhibitors of the invention is used to reduceor prevent hyperinsulinemia. In an embodiment of the invention, a ROCKinhibitor is used to treat diabetes (particularly type 2 diabetes). ROCKinhibitors of the invention may also be used to promote or restoreinsulin-mediated relaxation of vascular smooth muscle cells (VSMCs).

Angiogenesis

The invention provides methods and compounds for treating diseases anddisorders with an angiogenic component. According to the invention, incertain embodiments, such diseases and disorders are treated byadministering to a subject an effective amount of a ROCK inhibitor.According to the invention, such diseases and disorders can also betreated by administering an effective amount of a rho kinase inhibitorand an effective amount of an angiogenesis inhibitor. According to theinvention, ocular diseases and disorders having an angiogenic componentare treated in this manner. In one embodiment, the invention provides amethod of treating age related macular degeneration (AMD), which occursin “dry” and “wet” forms. The “wet” form of AMD causes vision loss dueto abnormal blood vessel growth (neovascularization). Bleeding, leaking,and scarring from these retinal blood vessels eventually causesirreversible damage to the photoreceptors. The dry form results fromatrophy of the retinal pigment epithelial layer, which causes visionloss through loss of photoreceptors (rods and cones) in the central partof the eye. In another embodiment, the invention provides a method oftreating choroidal neovascularization (CNV). Choroidalneovascularization is a process in which new blood vessels grow in thechoroid, through the Bruch membrane and invade the subretinal space, andis a symptom of, among other causes, age-related macular degeneration,myopia and ocular trauma. In another embodiment, the invention providesa method of treating diabetic macular edema (DME). In anotherembodiment, the invention provides a method of treating macular edemathat is secondary to branch retinal vein occlusion (BRVO) or centralretinal vein occlusion (CRVO). In other embodiments, the diseases to betreated include, without limitation, retinal neovascularization,infectious and non-infectious, corneal neovascularization infectious andnon-infectious, iris neovascularization, uveitis, neovascular glaucoma,and retinitis of prematurity (ROP). The method of treatment can beprophylactic, such as to stave off corneal neovascularization aftercorneal transplant, or to modulate the wound healing process intrabeculectomy surgery. These diseases and disorders may becharacterized as having an angiogenic component. According to theinvention, such disorders are treated by administering a ROCK inhibitor,and an angiogenesis inhibitor.

Accordingly, in one such embodiment, the disease or disorder is AMD, anda subject in need of treatment for AMD is administered an amount of aROCK inhibitor effective to treat AMD. In another embodiment, thesubject is administered a ROCK inhibitor and an angiogenesis inhibitorin amounts effective to treat AMD. In some embodiments, the angiogenesisinhibitor is a VEGFR2 antagonist. In certain such embodiments, theVEGFR2 antagonist binds to VEGF. In other such embodiments, the VEGFR2antagonist binds to VEGFR2. Such VEGFR2-binding inhibitors includeagents that bind to the extracellular domain of VEGFR2, including butnot limited to antibodies and VEGFR2-binding fragments thereof, andagents that interact with the intracellular domain of VEGFR2 and blockactivation of VEGFR2-dependent signal transduction. VEGFR2 antagonistsfurther include agents that interact with other cellular components toblock VEGFR2-dependent signal transduction. In other embodiments of theinvention, other ocular diseases and disorders having an angiogeniccomponent, such as are indicated above, are similarly treated.

According to the invention, a ROCK inhibitor and an angiogenesisinhibitor are administered to a subject in amounts effective amount totreat or preventing a pathologic condition characterized by excessiveangiogenesis. Such conditions, involving for example, vascularizationand/or inflammation, include atherosclerosis, rheumatoid arthritis (RA),hemangiomas, angiofibromas, and psoriasis. Other non-limiting examplesof angiogenic disease are retinopathy of prematurity (retrolentalfibroplastic), corneal graft rejection, corneal neovascularizationrelated to complications of refractive surgery, cornealneovascularization related to contact lens complications, cornealneovascularization related to pterygium and recurrent pterygium, cornealulcer disease, and non-specific ocular surface disease,insulin-dependent diabetes mellitus, multiple sclerosis, myastheniagravis, Chron's disease, autoimmune nephritis, primary biliarycirrhosis, acute pancreatitis, allograph rejection, allergicinflammation, contact dermatitis and delayed hypersensitivity reactions,inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis,cognition defects induced by neuronal inflammation, Osler-Webersyndrome, restenosis, and fungal, parasitic and viral infections,including cytomegaloviral infections.

The invention provides pan-ROCK inhibitors (i.e., compounds that inhibitROCK1 and ROCK2). One study observed that ROCK2 is frequently overexpressed in hepatocellular cancer compared to non-timorous livers whileROCK1 expression is unaltered. Other cancers that may benefit fromtreatment with a ROCK2 selective inhibitor include, but are not limitedto, colon and bladder cancer. In contrast, ROCK1 expression levels havebeen observed to be higher in mammary tumors. Any cancer may be testedto determine whether there is overexpression of ROCK1 and/or ROCK2 andtreated accordingly. In certain circumstances, ROCK 1 and ROCK2 isoformsshow similarity in regulating certain downstream targets and neitherisoform seems to be predominant. In such cases, a pan-ROCK inhibitor maybe preferred.

Combinations with Other Agents

Compounds of the invention can be advantageously administered withsecond agents to patients in need thereof. When ROCK inhibitor isadministered with a second agent, the ROCK inhibitor and the secondagent can be administered sequentially or concomitantly. Sequentiallymeans that one agent is administered for a time followed byadministration of the other agent, which may be followed byadministration of the first agent. When agents are administeredsequentially, the level of one agent may not be maintained at atherapeutically effective level when the second agent is administered,and vice versa. Concomitantly means that the first and second agents areadministered according to a schedule that maintains both agents at asubstantially therapeutically effective level, even though the agentsare not administered simultaneously. Each agent can be administered insingle or multiple doses, and the doses can be administered on anyschedule, including, without limitation, twice daily, daily, weekly,every two weeks, and monthly.

The invention also includes adjunctive administration. Adjunctiveadministration means that a second agent is administered to a patient inaddition to a first agent that is already being administered to treat adisease or disease symptom. In some embodiments, adjunctiveadministration involves administering a second agent to a patient inwhich administration of the first agent did not sufficiently treat adisease or disease symptom. In other embodiments, adjunctiveadministration involves administration of the second agent to a patientwhose disease has been effectively treated by administration of thefirst agent, with the expectation that the adjunctive treatment improvesthe outcome of the treatment. In some embodiments, the effect ofadministering the first and second agents is synergistic. In someembodiments, administration of the first and second agents prevents orlengthens the time until relapse, compared to administration of eitherof the agents alone. In some embodiments, administration of the firstand second agents allows for reduced dosage and/or frequency ofadministration of the first and second agent.

Anti-inflammatories and immunosuppressants that can be administered incombination with the compounds of the present invention include steroiddrugs such as glucocorticoids (e.g., dexamethasone), FK506 (tacrolimus),ciclosporin, fingolimod, interferon, such as IFNβ or IFNγ, a tumornecrosis factor-alpha (TNF-α) binding protein such as infliximab(Remicade), etanercept (Enbrel), or adalimumab (Humira), mycophenolicacid, MMF, Methotrexate, NSAID, Statins,Sirolimus/temsirolimus/everolimus, abatacept (Orencia), anakinra(Kineret), certolizumab (Cimzia), golimumab (Simponi), ixekizumab(Taltz), natalizumab (Tysabri), rituximab (Rituxan), secukinumab(Cosentyx), tocilizumab (Actemra), ustekinumab (Stelara), vedolizumab(Entyvio), basiliximab (Simulect), daclizumab (Zinbryta), muromonab(Orthoclone OKT3), Jakafi (Ruxolitinib), Xeljanz (Tofacitnib), andOtezla (Apremilast).

In an embodiment of the invention, a rho-kinase inhibitor of theinvention and an anti-neoplastic agent are administered to a subject inneed thereof. In another embodiment, a rho-kinase inhibitor of theinvention and an angiogenesis inhibitor are administered to a subject inneed thereof. In another embodiment, a rho-kinase inhibitor of theinvention and an anti-inflammatory agent are administered to a subjectin need thereof. In yet another embodiment, a ROCK inhibitor of theinvention and an immunosuppressant are administered. The second agentcan be, without limitation, a small molecule, an antibody or antigenbinding fragment thereof, or radiation.

Antineoplastic agents include, without limitation, cytotoxicchemotherapeutic agents, targeted small molecules and biologicalmolecules, and radiation. Compounds and agents that can be administeredfor oncological treatment, in addition to a rho kinase inhibitor of theinvention, include the following: irinotecan, etoposide, camptothecin,5-fluorouracil, hydroxyurea, tamoxifen, paclitaxel, capcitabine,carboplatin, cisplatin, bleomycin, dactomycin, gemcitabine, doxorubicin,danorubicin, cyclophosphamide, and radiotherapy, which can be external(e.g., external beam radiation therapy (EBRT)) or internal (e.g.,brachytherapy (BT)).

Targeted small molecules and biological molecules include, withoutlimitation, inhibitors of components of signal transduction pathways,such as modulators of tyrosine kinases and inhibitors of receptortyrosine kinases, and agents that bind to tumor-specfic antigens.Examples include inhibitors of epidermal growth factor receptor (EGFR),including gefitinib, erlotinib, and cetuximab, inhibitors of HER2 (e.g.,trastuzumab, trastuzumab emtansine (trastuzumab-DM1; T-DM1) andpertuzumab), anti-VEGF antibodies and fragments (e.g., bevacizumab),antibodies that inhibit CD20 (e.g., rituximab, ibritumomab), anti-VEGFRantibodies (e.g., ramucirumab (IMC-1121B), IMC-1C11, and CDP791),anti-PDGFR antibodies, and imatinib. Small molecule kinase inhibitorscan be specific for a particular tyrosine kinase or be inhibitors of twoor more kinases. For example, the compoundN-(3,4-dichloro-2-fluorophenyl)-7-({[(3aR,6aS)-2-methyloctahydrocyclopenta[c]pyrrol-5-yl]methyl}oxy)-6-(methyloxy)quinazolin-4-amine (also known asXL647, EXEL-7647 and KD-019) is an in vitro inhibitor of severalreceptor tyrosine kinases (RTKs), including EGFR, EphB4, KDR (VEGFR),Flt4 (VEGFR3) and ErbB2, and is also an inhibitor of the SRC kinase,which is involved in pathways that result in nonresponsiveness of tumorsto certain TKIs. In an embodiment of the invention, treatment of asubject in need comprises administration of a ROCK inhibitor of FormulasI-IX and administration of KD-019.

Dasatinib (BMS-354825; Bristol-Myers Squibb, New York) is another orallybioavailable, ATP-site competitive Src inhibitor. Dasatanib also targetsBcr-Abl (FDA-approved for use in patients with chronic myelogenousleukemia (CML) or Philadelphia chromosome positive (Ph+) acutelymphoblastic leukemia (ALL)) as well as c-Kit, PDGFR, c-FMS, EphA2, andSrc family kinases. Two other oral tyrosine kinase inhibitor of Src andBcr-Abl are bosutinib (SKI-606) and saracatinib (AZD0530).

According to the invention, angiogenesis inhibitors can be administeredto a subject in conjunction with compounds of the invention.Angiogenesis inhibitors include any substance that inhibits the growthof new blood vessels. For example, angiogenesis inhibitors includeantagonists of VEGF, P1GF, and VEGF receptors, including the antibodiesdisclosed herein. A VEGF antagonist reduces or blocks a function in acell that is associated with VEGF. A VEGF antagonist may act on VEGF, bybinding to VEGF and blocking binding to its receptors and/or may act onanother cellular component involved in VEGF-mediated signaltransduction. Similarly, a VEGFR2 antagonist is an agent that reduces orblocks VEGFR2-mediated signal transduction by binding to VEGFR2 andblocking ligand binding or interaction with a VEGFR2 substrate, or actson another cellular component to reduce or block VEGFR2-mediated signaltransduction. Thus, angiogenesis inhibitors include anti-VEGFR2antibodies, and antagonists of, without limitation, VEGF, VEGFR1,VEGFR2, PDGF, PDGFR-β, neuropilin-1 (NRP1), and complement.

Angiogenesis inhibitors include intracellular agents that block signaltransduction mediated by, for example, VEGF, PDGF, ligands of VEGF orPDGF receptors, or complement. Intracellular agents that inhibitangiogenesis inhibitors include the following, without limitation.Sunitinib (Sutent; SU11248) is a panspecific small-molecule inhibitor ofVEGFR1-VEGFR3, PDGFRα and PDGFRβ, stem cell factor receptor (cKIT),Flt-3, and colony-stimulating factor-1 receptor (CSF-1R). Axitinib(AG013736; Inlyta) is another small molecule tyrosine kinase inhibitorthat inhibits VEGFR-1-VEGFR-3, PDGFR, and cKIT. Cediranib (AZD2171) isan inhibitor of VEGFR-1-VEGFR-3, PDGFRO, and cKIT. Sorafenib (Nexavar)is another small molecular inhibitor of several tyrosine proteinkinases, including VEGFR, PDGFR, and Raf kinases. Pazopanib (Votrient;(GW786034) inhibits VEGFR-1, -2 and -3, cKIT and PDGFR. Foretinib(GSK1363089; XL880) inhibits VEGFR2 and MET. CP-547632 is as a potentinhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF)kinases. E-3810 ((6-(7-((1-aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yloxy)-N-methyl-1-naphthamide) inhibitsVEGFR-1, -2, and -3 and FGFR-1 and -2 kinases in the nanomolar range.Brivanib (BMS-582664) is a VEGFR-2 inhibitor that also inhibits FGFreceptor signaling. CT-322 (Adnectin) is a small protein based on ahuman fibronectin domain and binds to and inhibits activation of VEGFR2.Vandetanib (Caprelas; Zactima; ZD6474) is an inhibitor of VEGFR2, EGFR,and RET tyrosine kinases. X-82 (Xcovery) is a small molecule indolinoneinhibitor of signaling through the growth factor receptors VEGFR andPDGFR

Pharmaceutical Compositions

In one aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds of Formulas I-IX, formulatedtogether with one or more pharmaceutically excipients. As describedbelow, the pharmaceutical compositions of the present invention may bespecially formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; (2) parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation; (3)topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals with toxicity, irritation,allergic response, or other problems or complications, commensurate witha reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like (see, e.g., Berge et al. (1977) “PharmaceuticalSalts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like(see, e.g., Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges, powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable excipients including apharmaceutically-acceptable carrier, such as sodium citrate or dicalciumphosphate, and/or any of the following: (1) fillers or extenders, suchas starches, lactose, sucrose, glucose, mannitol, and/or silicic acid;(2) binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants,such as glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds andsurfactants, such as poloxamer and sodium lauryl sulfate; (7) wettingagents, such as, for example, cetyl alcohol, glycerol monostearate, andnon-ionic surfactants; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, zincstearate, sodium stearate, stearic acid, and mixtures thereof; (10)coloring agents; and (11) controlled release agents such as crospovidoneor ethyl cellulose. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-shelled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides diluents, the oral compositions can also include additionalexcipients such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain additional excipients such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms upon the subject compoundsmay be ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

Routes of Administration and Dose

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

In certain embodiments, a dose of a compound or a composition isadministered to a subject every day, every other day, every couple ofdays, every third day, once a week, twice a week, three times a week, oronce every two weeks. If desired, the effective daily dose of the activecompound may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms. In some embodiments, adose(s) of a compound or a composition is administered for 2 days, 3days, 5 days, 7 days, 14 days, or 21 days. In certain embodiments, adose of a compound or a composition is administered for 1 month, 1.5months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months ormore.

The above-described administration schedules are provided forillustrative purposes only and should not be considered limiting. Aperson of ordinary skill in the art will readily understand that alldoses are within the scope of the invention.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compounds for use in the methods of the invention can beadministered as such or in admixtures with pharmaceutically acceptablecarriers and can also be administered in conjunction with antimicrobialagents such as penicillins, cephalosporins, aminoglycosides andglycopeptides. Conjunctive therapy thus includes sequential,simultaneous and separate administration of the active compound in a waythat the therapeutic effects of the first administered one is notentirely disappeared when the subsequent is administered.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”,W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” O and B books, Corvallis, Ore., U.S.A., 1977).

Microemulsification technology may be employed to improvebioavailability of lipophilic (water insoluble) pharmaceutical agents.Examples include Trimetrine (Dordunoo, S. K., et al., Drug Developmentand Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen,P. C., et al., J Pharm Sci 80(7), 712-714, 1991). Among other things,microemulsification provides enhanced bioavailability by preferentiallydirecting absorption to the lymphatic system instead of the circulatorysystem, which thereby bypasses the liver, and prevents destruction ofthe compounds in the hepatobiliary circulation.

Controlled Release

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. Release can be manipulatedto be pH dependent, for example, using a pH sensitive coating thatreleases only at a low pH, as in the stomach, or a higher pH, as in theintestine. An enteric coating can be used to prevent release fromoccurring until after passage through the stomach. Multiple coatings ormixtures of cyanamide encapsulated in different materials can be used toobtain an initial release in the stomach, followed by later release inthe intestine. Release can also be manipulated by inclusion of salts orpore forming agents, which can increase water uptake or release of drugby diffusion from the capsule. Excipients that modify the solubility ofthe drug can also be used to control the release rate. Agents whichenhance degradation of the matrix or release from the matrix can also beincorporated. They can be added to the drug, added as a separate phase(i.e., as particulates), or can be co-dissolved in the polymer phasedepending on the compound. Types of degradation enhancers includeinorganic salts such as ammonium sulfate and ammonium chloride, organicacids such as citric acid, benzoic acid, and ascorbic acid, inorganicbases such as sodium carbonate, potassium carbonate, calcium carbonate,zinc carbonate, and zinc hydroxide, and organic bases such as protaminesulfate, spermine, choline, ethanolamine, diethanolamine, andtriethanolamine and surfactants such as Tween® and Pluronic®. Poreforming agents which add microstructure to the matrices (i.e., watersoluble compounds such as inorganic salts and sugars) are added asparticulates. The range should be between one and thirty percent (w/wpolymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

It is to be understood and expected that variations in the principles ofinvention herein disclosed can be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention. The following Examples further illustrate theinvention, but should not be construed to limit the scope of theinvention in any way. All references cited herein are herebyincorporated by reference in their entirety.

EXAMPLES Example 1

All solvents and reagents were obtained commercially and used asreceived. ¹H NMR spectra were recorded on a Bruker instrument (300 MHzor 400 MHz) in the cited deuterated solvents. Chemical shifts are givenin ppm, and coupling constants are in hertz. All final compounds werepurified by flash chromatography using 220-400 mesh silica gel orreverse-phase HPLC with CH₃CN/water as the solvents. Thin-layerchromatography was done on silica gel 60 F-254 (0.25-nm thickness)plates. Visualization was accomplished with UV light and/or 10%phosphomolybdic acid in ethanol. Nominal (low resolution) mass spectrawere acquired on either a Waters LCT or an Applied Biosystems API 3000mass spectrometer. High resolution mass spectra (HRMS) were acquired oneither a Waters LCT or an Agilent TOF mass spectrometer. All other LC-MSexperiments were done on an Agilent 1100 HPLC coupled with an Agilentsingle quadrupole mass spectrometer. Compound purity was determined by aLC-MS with 230 nM and 254 nM wavelengths. All final compounds reportedhere have purity ≥95%.

General Procedure A

EDCI coupling: compound of general structure 1 (1 equiv), EDCI (1equiv), HOBt (0.2 equiv) and DIEA (2 equiv) were dissolved in DMFstirred at 25° C. for 5 min. Then to the mixture was added amine (1equiv). The mixture was stirred at 25° C. for 16 hr. The mixture waspoured into water and extracted with EtOAc. Combined organic phases werewashed with brine, dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography to afford compound of general structure 2.

HATU coupling: compound of general structure 1 (1 equiv), HATU (1.25equiv) and DIPEA (1.5 equiv) were dissolved in DMF stirred at 23° C. for15 minutes. 1H-indazol-5-amine (1 equiv) was introduced to the reactionmixture and solution continued to stir at 23° C. for another 16 hours.Reaction mixture was diluted with water and extracted with EtOAc.Combined organic layers were washed with brine, dried over Na₂SO₄,filtered and concentrated under reduced pressure to give crude material,which was purified on silica gel to afford the desired compound ofgeneral structure 2.

Boc-deprotection: compound of general structure 2 was dissolved in DCMand 4N HCl in dioxane was introduced to the solution. Reaction mixturewas stirred at 23° C. for 1 hour. Reaction mixture was concentratedunder reduced pressure to give a crude reaction mixture, which waspurified via reverse phase preparative HPLC to afford the desiredcompound of general structure 3.

Benzyl group removal: compound of general structure 2 (1 equiv) andconc. HCl (1.2 equiv) were dissolved in MeOH and 10% dry Pd/C added.Reaction mixture was stirred under an atmosphere of H₂ (1 atm) at 50° C.for 5 hours. The reaction mixture was filtered and the filtrateconcentrated under reduced pressure to give the crude product which waspurified via reverse phase preparative HPLC to afford the desiredcompound of general structure 3.

General Procedure B

Amine HCl salt (2 equiv) was dissolved in methanol and tryethylamineadded (2 equiv). The reaction solution was stirred for 15 min afterwhich ketone or aldehyde of general structure 4 (1 equiv) along withHOAc (4 equiv) were added. The stirring was continued for another 15 minand NaBH₃CN added. The reaction temperature was increased to 60° C. andstirring continued for 16 hours. Reaction mixture was diluted with waterand extracted with EtOAc. Combined organic layers were washed withbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure to give crude material, which was purified on silica gel toafford the desired compound of general structure 5.

K₂CO₃ (2 equiv), LiI (0.05 equiv) and benzylbromide were added to thesolution of compound of general structure 5 (1 equiv) in MeCN at roomtemperature. Reaction temperature was elevated to 60° C. and stirringcontinued for 16 hours. Reaction mixture was diluted with water andextracted with EtOAc. Combined organic layers were washed with brine,dried over Na₂SO₄, filtered and concentrated under reduced pressure togive crude material, which was purified on silica gel to afford thedesired compound of general structure 6.

Solution of compound of general structure 6 (1 equiv), Pd(PPh₃)₄ (0.15equiv)), K₃PO₄ (3 equiv) in THF was purged with nitrogen for fiveminutes at room temperature. Reaction temperature was elevated to 120°C. and stirring continued for 48 hours. Solvent was removed underreduced pressure and residue purified via silica gel chromatography toafford the desired compound of general structure 7.

Compound of general structure 7 (1 equiv) was dissolved in methanolwater mixture and NaOH (2 equiv) added. Reaction continued to stir at30° C. for 16 hours. Reaction solution was acidified to pH=5.Purification of the crude residue was accomplished by reverse phasepreparative HPLC to afford the desire compound of general structure 8.

Compound of general structure 8 (1 equiv), HATU (1.25 equiv) and DIPEA(1.5 equiv) were dissolved in DMF stirred at 23° C. for 15 minutes.1H-indazol-5-amine (1 equiv) was introduced to the reaction mixture andsolution continued to stirr at 23° C. for another 16 hours. Reactionmixture was diluted with water and extracted with EtOAc. Combinedorganic layers were washed with brine, dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give crude material, which waspurified on silica gel to afford the desired compound of generalstructure 9.

Compound of general structure 9 (1 equiv) and conc. HCl (1.2 equiv) weredissolved in MeOH and 10% dry Pd/C added. Reaction mixture was stirredunder an atmosphere of H₂ (1 atm) at 50° C. for 5 hours. The reactionmixture was filtered and the filtrate concentrated under reducedpressure to give the crude product, which was purified via reverse phasepreparative HPLC to afford the desired compound of general structure 10.

General Procedure C

Compound of general structure 11 (1 equiv) was dissolved in acetic acidand PtO₂ (0.1 equiv) added. Reaction solution was stirred under anatmosphere of hydrogen (1 atm) at room temperature for 16 hours. Theresulting solution was filtered off and concentrated under reducedpressure. The residue was basified with 2N NaOH to pH=9 and mixtureextracted with EtOAc. Combined organic layers were washed with brine,dried over Na₂SO₄, filtered and concentrated under reduced pressure togive crude material, which was purified on silica gel to afford thedesired compound of general structure 12.

Compound of general structure 12 (1 equiv) was dissolved in DCM andBoc₂O (1.5 equiv), DIPEA (2 equiv) were added. Reaction continued tostir at room temperature for 16 hours. Reaction mixture was diluted withwater and extracted with EtOAc. Combined organic layers were washed withbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure to give crude material, which was purified on silica gel toafford the desired compound of general structure 13.

Compound of general structure 13 (1 equiv) was dissolved in methanolwater mixture and NaOH (2 equiv) added. Reaction continued to stir at30′C for 16 hours. Reaction solution was acidified to pH=5. Purificationof the crude residue was accomplished by reverse phase preparative HPLCto afford the desire compound of general structure 14.

Compound of general structure 14 (1 equiv), HATU (1.25 equiv) and DIPEA(1.5 equiv) were dissolved in DMF stirred at 23° C. for 15 minutes.1H-indazol-5-amine (1 equiv) was introduced to the reaction mixture andsolution continued to stir at 23° C. for another 16 hours. Reactionmixture was diluted with water and extracted with EtOAc. Combinedorganic layers were washed with brine, dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give crude material, which waspurified on silica gel to afford the desired compound of generalstructure 15.

Compound of general structure 15 was dissolved in DCM and 4N HCl indioxane was introduced to the solution. Reaction mixture was stirred at23° C. for 1 hour. Reaction mixture was concentrated under reducedpressure to give a crude reaction mixture which was purified via reversephase preparative HPLC to afford the desired compound of generalstructure 16.

General Procedure D

A solution of compound of general structure 17 (1 equiv) and aldehyde orketone (1 equiv) in MeOH (3.00 mL) was stirred at 23° C. for 16 hr.After 16 hours NaBH₃CN (1.2 equiv) was added and mixture stirred foradditional 3 hours. Reaction mixture was quenched by the addition ofwater and resulting mixture extracted with EtOAc. Combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure. Crude material was purified by preparative HPLC to affordcompound of general structure 18.

General Procedure E

Compound of general structure 19 (1 equiv) was dissolved in CCl₄ andAIBN (0.1 equiv) followed by NBS (1.2 equiv) were added at roomtemperature. The reaction temperature was raised to 60° C. and stirringcontinued for 16 hours. Reaction mixture was filtered off and solventremoved under reduced pressure. Crude material 20 was used in the nextstep without additional purification.

Compound of general structure 20 (1 equiv) and alkyl amine (2 equiv) inTHE was stirred at 70° C. for 16 hours. The reaction mixture wasconcentrated to under reduced pressure and material purified via silicagel chromatography to afford a compound of general structure 21.

NaOH (2 equiv) was introduced to a reaction vessel containing compoundof general structure 21 (1 equiv) dissolved in MeOH/H₂O mixture.Reaction was stirred at 20° C. for 16 hours. Solvent was removed underreduce pressure. The crude residue was dissolved in water (10 mL) andneutralized with 6 N HCl carefully until pH=8. The suspension wasfiltered and solids were collected and dried to afford compound ofgeneral structure 22.

Compound general of structure 22 (1 equiv), HATU (1.25 equiv) and DIPEA(2 equiv) were dissolved in DMF and stirred at 20° C. for 15 minutes.1H-indazol-5-amine (1 equiv) was introduced to the reaction mixture andstirred at 20° C. for 15.8 hours. Reaction diluted with water andextracted with EtOAc. Combined organic layers were washed with brine (15mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure. Crude reaction mixture purified via reverse phase preparativeHPLC to afford compound of general structure 23.

General Procedure F

Benzyl amine (1 equiv) was dissolved in dichloromethane. Thecorresponding aryl boronic acid 24 (1 equiv) was added to the reactionsolution followed by glyoxylic acid (1 equiv) under an atmosphere ofnitrogen. The reaction mixture was warmed up to 40° C. and temperaturemaintained for 16 hours. Reaction mixture was concentrated under reducedpressure. Crude reaction mixture purified via reverse phase preparativeHPLC to afford compound of general structure 25.

Compound general of structure 25 (1 equiv), HATU (1.25 equiv) and DIPEA(2 equiv) were dissolved in DMF and stirred at 20° C. for 15 minutes.1H-indazol-5-amine (1 equiv) was introduced to the reaction mixture andstirred at 20° C. for 16 hours. Reaction diluted with water andextracted with EtOAc. Combined organic layers were washed with brine,dried over Na₂SO₄, filtered and concentrated under reduced pressure.Crude reaction mixture purified via normal phase silica gelchromatography to afford compound of general structure 26.

Compound of general structure 26 (1 equiv) and conc. HCl (1.2 equiv)were dissolved in MeOH and 10% dry Pd/C added. Reaction mixture wasstirred under an atmosphere of H2 (1 atm) at 60° C. for 3 hours. Thereaction mixture was filtered and the filtrate concentrated underreduced pressure to give the crude product, which was purified viareverse phase preparative HPLC to afford the desired compound of generalstructure 27.

Example 2 N-(1H-indazol-5-yl)-2-(methylamino)-2-phenylacetamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-phenylacetamide as an off-whitesolid (80%). ¹H NMR (400 MHz, CD₃OD) δ 8.34 (s, 1H), 8.27 (s, 1H),7.68-7.65 (m, 2H), 7.60-7.53 (m, 5H), 5.1 (s, 1H), 2.69 (s, 3H). MS(ES+) m/e 281.1 (M+H).

Example 3 N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroquinoline-3-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-1,2,3,4-tetrahydroquinoline-3-carboxamide as ayellow solid (23%). ¹H NMR (400 MHz, MeOD-d₃) δ 13.75 (s, 1H), 10.84 (s,1H), 8.94 (s, 1H), 8.80 (s, 1H), 8.26 (m, 2H), 7.71 (m, 2H), 7.28 (m,2H), 6.63 (s, 1H), 4.21 (m, 1H), 3.97 (m, 1H), 3.65 (m, 3H). MS (ES+)m/e 293.1 (M+H)⁺.

Example 4N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide as ayellow solid (51%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (brs, 1H), 9.96(s, 1H), 8.21 (s, 1H), 8.18 (s, 1H), 8.03 (s, 1H), 7.53-7.48 (m, 2H),7.20-7.12 (m, 3H), 7.10-7.08 (m, 1H), 4.10-3.93 (m, 3H), 3.75-3.64 (m,1H), 3.07-3.02 (m, 1H), 2.93-2.91 (m, 1H). MS (ES+) m/e 293.0 (M+H)⁺.

Example 5N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-1-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-1-carboxamide as awhite solid (18%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.16 (s,1H), 8.21 (s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.52-7.46 (m, 2H),7.37-7.35 (m, 1H), 7.18-7.13 (m, 3H), 4.64 (d, J=4.0 Hz, 1H), 3.25-3.22(m, 1H), 2.97-2.81 (m, 2H), 2.75-2.65 (m, 1H). MS (ES+) m/e 293.1(M+H)⁺.

Example 6 N-(1H-indazol-5-yl)isoindoline-1-carboxamide

The reaction was conducted following general protocol B. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)isoindoline-1-carboxamide as a white solid (10%). ¹HNMR (400 MHz, DMSO-d₆) δ 12.99 (brs, 1H), 10.09 (s, 1H), 8.18 (s, 1H),8.01 (s, 1H), 7.56-7.44 (m, 3H), 7.34-7.20 (m, 3H), 4.96 (brs, 1H),4.40-4.26 (m, 2H), 3.79 (brs, 1H). MS (ES+) m/e 279.1 (M+H)⁺.

Example 7N-(2-(dimethylamino)ethyl)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(2-(dimethylamino)ethyl)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamideas a white solid (50%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.28 (brs, 1H),8.07 (brs, 1H), 7.78-6.91 (m, 5H), 6.54 (d, J=7.6 Hz, 1H), 6.48 (s, 1H),4.01-3.67 (m, 3H), 3.61 (s, 3H), 2.32-2.11 (m, 3H), 2.11-2.10 (m, 9H).MS (ES+) m/e 382.1 (M+H)⁺.

Example 8N-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide

The reaction was conducted following general protocol C. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxamide as alight brown solid (46%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.01 (brs, 1H),10.59 (s, 1H), 8.23 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.50-7.48 (m,1H), 7.44-7.42 (m, 1H), 7.29-7.22 (m, 1H), 7.20-7.13 (m, 3H), 4.05-4.00(m, 1H), 3.95-3.85 (m, 1H), 3.79-3.75 (m, 1H), 3.37-3.33 (m, 1H),3.22-3.19 (m, 1H). MS (ES+) m/e 293.0 (M+H)+.

Example 9 N-(1H-indazol-5-yl)-3-methylisoindoline-1-carboxamide

The reaction was conducted following general protocol B. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-3-methylisoindoline-1-carboxamide as a white solid(29%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (brs, 1H), 10.04 (s, 1H), 8.15(s, 1H), 8.02 (s, 1H), 7.53-7.42 (m, 3H), 7.35-7.21 (m, 3H), 4.89 (s,1H), 4.58-4.57 (m, 1H), 4.01 (s, 1H), 1.47 (d, J=6.4 Hz, 3H). MS (ES+)m/e 293.0 (M+H)+.

Example 10 N-(1H-indazol-5-yl)pyrrolidine-2-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)pyrrolidine-2-carboxamide as a white solid (33%). MS(ES+) m/e 231.1 (M+H)+.

Example 11 N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)pyrrolidine-3-carboxamide as a white solid (41%). MS(ES+) m/e 231.1 (M+H)⁺.

Example 12 (2S,5R)-N-(1H-indazol-5-yl)-5-phenylpyrrolidine-2-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to afford(2S,5R)-N-(1H-indazol-5-yl)-5-phenylpyrrolidine-2-carboxamidecarboxamide as a white solid (26%). MS (ES+) m/e 307.0 (M+H)⁺.

Example 13 N-(1H-indazol-5-yl)piperidine-2-carboxamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)piperidine-2-carboxamide as a white solid (39%). MS(ES+) m/e 245.0 (M+H)⁺.

Example 14N-(1H-indazol-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(4-(4-methoxyphenoxy)phenyl)-2-(methylamino)acetamideas a white solid (7%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.05(s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.47 (s, 2H), 7.45 (d, J=8.8 Hz,2H), 7.00-6.93 (m, 4H), 6.89 (d, J=8.4 Hz, 2H), 4.20 (d, J=7.2 Hz, 1H),3.75 (s, 3H), 2.60 (m, 1H), 2.30 (d, J=4.8 Hz, 3H). MS (ES+) m/e 403.1(M+H)⁺.

Example 15N-(1H-indazol-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol A. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(4-(3-methoxyphenoxy)phenyl)-2-(methylamino)acetamideas a white solid (8%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.98 (s, 1H), 10.08(s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.51-7.48 (m, 4H), 7.27 (t, J=8.4Hz, 1H), 7.00 (d, J=8.4 Hz, 2H), 6.72 (dd, J=8.4, 2.0 Hz, 1H), 6.58 (t,J=2.4 Hz, 1H), 6.53 (dd, J=8.0, 2.4 Hz, 1H), 4.23 (s, 1H), 3.73 (s, 3H),2.31 (s, 3H). MS (ES+) m/e 403.1 (M+H)⁺.

Example 162-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as awhite solid (7%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.98 (s, 1H), 10.10 (s,1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.52-7.40 (m, 6H), 4.24 (s, 1H), 2.72(m, 1H), 2.28 (s, 3H). MS (ES+) m/e 315.0 (M+H)⁺.

Example 172-(4-chlorophenyl)-2-(ethylamino)-N-(1H-indazol-5-yl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as awhite solid (12%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.13 (s,1H), 8.28 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.52 (d, J=8.4 Hz, 2H),7.49-7.39 (m, 4H), 4.39 (s, 1H), 2.51-2.50 (m, 2H), 1.08 (t, J=6.4 Hz,3H). MS (ES+) m/e 329.1 (M+H)⁺.

Example 182-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-chlorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide as awhite solid (49%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.18 (s,1H), 8.21 (s, 1H), 8.09 (d, J=1.2 Hz, 1H), 8.00 (d, J=0.8 Hz, 1H),7.52-7.45 (m, 3H), 7.44-7.39 (m, 3H), 4.51 (s, 1H), 2.70-2.66 (m, 1H),1.03 (t, J=6.4 Hz, 1H). MS (ES+) m/e 343.1 (M+H)⁺.

Example 192-(4-fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as awhite solid (11%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.08 (s, 1H), 10.64 (s,1H), 9.59-9.31 (m, 2H), 8.07 (d, J=9.2 Hz, 2H), 7.67-7.64 (m, 2H), 7.52(d, J=8.8 Hz, 1H), 7.41-7.34 (m, 3H), 5.04 (s, 1H), 2.52 (s, 3H). MS(ES+) m/e 299.0 (M+H)⁺.

Example 202-(4-fluorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-fluorophenyl)-N-(1H-indazol-5-yl)-2-(isopropylamino)acetamide as awhite solid (54%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.17 (s,1H), 8.20 (s, 1H), 8.10 (d, J=1.2 Hz, 1H), 8.00 (d, J=0.8 Hz, 1H), 7.52(dd, J=8.4, 5.6 Hz, 2H), 7.49-7.39 (m, 2H), 7.17 (t, J=8.8 Hz, 2H), 4.51(s, 1H), 2.72-2.66 (m, 1H), 1.04 (t, J=6.8 Hz, 6H). MS (ES+) m/e 327.1(M+H)⁺.

Example 21 2-(ethylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(ethylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide as a white solid(39%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.00 (s, 1H), 10.17 (s, 1H), 8.23(s, 1H), 8.11 (s, 1H), 8.00 (s, 1H), 7.52-7.45 (m, 4H), 7.35 (t, J=7.2Hz, 2H), 7.30-7.28 (m, 1H), 4.43 (s, 1H), 2.63-2.52 (m, 2H), 1.09 (t,J=7.2 Hz, 3H). MS (ES+) m/e 295.0 (M+H)⁺.

Example 22 N-(1H-indazol-5-yl)-2-(isopropylamino)-2-phenylacetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(isopropylamino)-2-phenylacetamide as a whitesolid (63%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.17 (s, 1H),8.20 (s, 1H), 8.11 (d, J=1.2 Hz, 1H), 8.00 (d, J=0.8 Hz, 1H), 7.52-7.41(m, 4H), 7.34 (t, J=7.2 Hz, 2H), 7.30-7.27 (m, 1H), 4.51 (s, 1H),2.72-2.69 (m, 1H), 1.04 (dd, J=7.6, 6.4 Hz, 6H). MS (ES+) m/e 309.1(M+H)⁺.

Example 232-(ethylamino)-2-(4-fluorophenyl)-N-(1H-indazol-5-yl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(ethylamino)-2-(4-fluorophenyl)-N-(1H-indazol-5-yl)acetamide as awhite solid (8%). ¹H NMR (400 MHz, CD₃OD) δ 8.45 (s, 1H), 8.09 (d, J=1.2Hz, 1H), 8.00 (s, 1H), 7.67-7.61 (m, 2H), 7.50 (d, J=8.8 Hz, 1H), 7.42(dd, J=8.8, 1.6 Hz, 1H), 7.26-7.19 (m, 2H), 4.82 (s, 1H), 2.96-2.88 (m,2H), 1.29 (t, J=7.2 Hz, 3H). MS (ES+) m/e 313.1 (M+H)⁺.

Example 24N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide as awhite solid (4%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.96 (s, 1H), 10.01 (s,1H), 8.12 (s, 1H), 7.99 (s, 1H), 7.46 (s, 2H), 7.39 (d, J=8.8 Hz, 2H),6.90 (d, J=8.8 Hz, 2H), 4.15 (s, 1H), 3.72 (s, 3H), 2.28 (s, 3H). MS(ES+) m/e 311.1 (M+H)⁺.

Example 25N-(1H-indazol-5-yl)-2-(isopropylamino)-2-(4-methoxyphenyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(4-methoxyphenyl)-2-(methylamino)acetamide as awhite solid (52%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.98 (s, 1H), 10.11 (s,1H), 8.19 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.48-7.39 (m, 4H), 6.90(d, J=8.4 Hz, 2H), 4.45 (s, 1H), 3.72 (s, 3H), 2.72-2.68 (m, 1H), 1.05(d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS (ES+) m/e 339.1 (M+H)⁺.

Example 262-(ethylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(ethylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide as awhite solid (5%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.10 (s,1H), 8.20 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.50-7.41 (m, 4H), 6.91(d, J=8.8 Hz, 2H), 4.37 (s, 1H), 3.73 (s, 1H), 2.60-2.53 (m, 2H), 2.27(s, 3H), 1.08 (t, J=7.2 Hz, 3H). MS (ES+) m/e 325.1 (M+H)⁺.

Example 272-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide asa white solid (10%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.95 (s, 1H), 10.07(s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 7.99 (d, J=0.8 Hz, 1H), 7.49-7.41(m, 2H), 7.39 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 4.39 (s, 1H),3.72 (s, 3H), 2.05-2.00 (m, 1H), 0.42-0.33 (m, 4H). MS (ES+) m/e 337.0(M+H)⁺.

Example 28 N-(1H-indazol-5-yl)-2-(methylamino)-2-(p-tolyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-(p-tolyl)acetamide as a whitesolid (7%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.04 (s, 1H),8.11 (s, 1H), 7.99 (s, 1H), 7.46 (s, 2H), 7.36 (d, J=8.0 Hz, 2H), 7.14(d, J=8.0 Hz, 2H), 4.19 (s, 1H), 2.28 (d, J=7.2 Hz, 6H). MS (ES+) m/e295.1 (M+H)⁺.

Example 29 N-(1H-indazol-5-yl)-2-(isopropylamino)-2-(p-tolyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(isopropylamino)-2-(p-tolyl)acetamide as a whitesolid (44%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.98 (s, 1H), 10.17 (s, 1H),8.17 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.48-7.37 (m, 4H), 7.16 (d,J=7.6 Hz, 2H), 4.51 (s, 1H), 2.76-2.70 (m, 1H), 2.27 (s, 3H), 1.05 (dd,J=8.4, 6.4 Hz, 6H). MS (ES+) m/e 323.1 (M+H)⁺.

Example 30 2-(ethylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(ethylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide as a white solid(13%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.08 (s, 1H), 8.24(s, 1H), 8.10 (s, 1H), 7.99 (s, 1H), 7.48-7.44 (m, 2H), 7.37 (d, J=8.0Hz, 2H), 7.15 (d, J=8.0 Hz, 2H), 4.34 (s, 1H), 2.59-2.51 (m, 2H), 2.27(s, 3H), 1.07 (t, J=7.2 Hz, 3H). MS (ES+) m/e 309.1 (M+H)⁺.

Example 31 2-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(cyclopropylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide as a whitesolid (23%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.96 (s, 1H), 10.07 (s, 1H),8.22 (s, 1H), 8.11 (s, 1H), 7.99 (s, 1H), 7.48-7.42 (m, 2H), 7.35 (d,J=8.0 Hz, 2H), 7.13 (d, J=8.0 Hz, 2H), 4.40 (s, 1H), 2.26 (s, 3H),2.07-1.99 (m, 1H), 0.42-0.29 (m, 4H). MS (ES+) m/e 321.1 (M+H)⁺.

Example 32N-(1H-indazol-5-yl)-2-(2′-methoxy-[1,1′-biphenyl]-4-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(2′-methoxy-[1,1′-biphenyl]-4-yl)-2-(methylamino)acetamideas a yellow solid (16%). ¹HNMR (400 MHz, DMSO-d₆) δ 13.00 (brs, 1H),10.13 (s, 2H), 8.28 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.53-7.43 (m,6H), 7.33-7.30 (m, 1H), 7.27 (dd, J=7.6, 2.0 Hz, 1H), 7.09 (d, J=7.6 Hz,1H), 7.01 (t, J=6.4 Hz, 1H), 4.27 (s, 1H), 2.34 (s, 3H). MS (ES+) m/e387.1 (M+H)⁺.

Example 332-(2′-fluoro-[1,1′-biphenyl]-4-yl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-(2′-fluoro-[1,1′-biphenyl]-4-yl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamideas a white solid (10%). ¹H NMR (400 MHz, DMSO-d₆) □ 12.98 (s, 1H), 10.14(s, 1H), 8.31 (s, 2H), 8.14 (s, 1H), 8.00 (s, 1H), 7.60 (d, J=8.4 Hz,2H), 7.54-7.48 (m, 5H), 7.42-7.38 (m, 1H), 7.33-7.26 (m, 2H), 4.30 (s,1H), 2.33 (s, 3H). MS (ES+) m/e 375.1 (M+H)⁺.

Example 342-((2-(dimethylamino)ethyl)amino)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to afford2-((2-(dimethylamino)ethyl)amino)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)acetamideas a white solid (27%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.01 (brs, 1H),10.19 (s, 1H), 8.26 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.49-7.43 (m,2H), 7.27 (t, J=7.8 Hz, 1H), 7.07-7.04 (m, 2H), 6.86 (dd, J=8.0, 2.0 Hz,1H), 4.36 (s, 1H), 3.75 (s, 3H), 2.65-2.57 (m, 2H), 2.55-2.53 (m, 2H),2.25 (s, 6H). MS (ES+) m/e 368.1 (M+H)⁺.

Example 35N-(1H-indazol-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol D. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(2-methoxyphenyl)-2-(methylamino)acetamide as apink solid (10%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.03 (brs, 1H), 9.99(brs, 1H), 8.20 (s, 2H), 8.13 (s, 1H), 8.01 (s, 1H), 7.52-7.46 (m, 2H),7.40 (dd, J=7.6, 1.6 Hz, 1H), 7.32-7.27 (m, 1H), 7.03 (d, J=8.0 Hz, 1H),6.96 (t, J=7.2 Hz, 1H), 4.59 (s, 1H), 3.83 (s, 3H), 2.34 (s, 3H). MS(ES+) m/e 311.0 (M+H)⁺.

Example 362-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to afford2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(4-methoxyphenyl)acetamide asalight brown solid (35%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.69 (s, 1H),9.14 (m, 2H), 8.06 (s, 2H), 7.60 (d, J=8.8 Hz, 2H), 7.53 (d, J=8.8 Hz,1H), 7.34 (dd, J=8.8, 2.0 Hz, 1H), 7.07 (d, J=8.8 Hz, 2H), 5.20-5.18 (m,1H), 3.78 (s, 3H), 1.32 (s, 9H). MS (ES+) m/e 353.1 (M+H)⁺.

Example 37 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to afford2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-phenylacetamide as a purplesolid (4%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.26 (s, 1H),8.35 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.49-7.46 (m, 4H), 7.32 (t,J=7.6 Hz, 2H), 7.26-7.22 (m, 1H), 4.50 (s, 1H), 1.10 (s, 9H). MS (ES+)m/e 323.1 (M+H)⁺.

Example 38 2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to afford2-(tert-butylamino)-N-(1H-indazol-5-yl)-2-(p-tolyl)acetamide as a whitesolid (12%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.98 (s, 1H), 10.20 (s, 1H),8.09 (s, 1H), 8.00 (s, 1H), 7.49-7.43 (m, 2H), 7.34 (d, J=8.0 Hz, 2H),7.12 (d, J=8.0 Hz, 2H), 4.44 (s, 1H), 2.26 (s, 3H), 1.09 (s, 9H). MS(ES+) m/e 337.1 (M+H)⁺.

Example 39 N-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-3-yl)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-3-yl)acetamide as a whitesolid (9%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.02 (brs, 1H), 10.21 (s, 1H),8.69 (d, J=1.6 Hz, 1H), 8.50 (dd, J=4.8, 1.6 Hz, 1H), 8.24 (s, 1H), 8.12(s, 1H), 8.01 (s, 1H), 7.90-7.88 (m, 1H), 7.50-7.45 (m, 2H), 7.41-7.39(m, 1H), 4.34 (s, 1H), 2.32 (s, 3H). MS (ES+) m/e 282.0 (M+H)⁺.

Example 40 N-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-2-yl)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-(pyridin-2-yl)acetamide as anoff-white solid (15%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.98 (brs, 1H),10.19 (s, 1H), 8.54 (dd, J=4.8, 0.8 Hz, 1H), 8.21 (s, 1H), 8.14 (d,J=1.2 Hz, 1H), 8.00 (s, 1H), 7.84-7.79 (m, 1H), 7.60 (d, J=8.0 Hz, 1H),7.48 (s, 2H), 7.34-7.30 (m, 1H), 4.44 (s, 1H), 2.36 (s, 3H). MS (ES+)m/e 282.0 (M+H)⁺.

Example 41N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide as anoff-white solid (42%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.11(s, 1H), 8.23 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.48-7.44 (m, 2H),7.27 (t, J=8.0 Hz, 1H), 7.11-7.04 (m, 2H), 6.88-6.83 (m, 1H), 4.26 (s,1H), 3.76 (s, 3H), 2.31 (s, 3H). MS (ES+) m/e 311.0 (M+H)⁺.

Example 422-(3-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to afford2-(3-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as anoff-white solid (63%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.00 (brs, 1H),10.16 (brs, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.59 (s, 1H),7.51-7.43 (m, 3H), 7.42-7.31 (m, 2H), 4.31 (s, 1H), 2.30 (s, 3H). MS(ES+) m/e 315.0 (M+H)⁺.

Example 432-(4-ethoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-ethoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as anoff-white solid (59%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.02 (brs, 1H),10.11 (brs, 1H), 8.23 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.49-7.43 (m,2H), 7.40 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 4.26 (s, 1H), 4.00(q, J=6.8 Hz, 2H), 2.31 (s, 3H), 1.31 (t, J=6.8 Hz, 3H). MS (ES+) m/e324.1 (M+H)⁺.

Example 44N-(1H-indazol-5-yl)-2-(4-isopropoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(4-isopropoxyphenyl)-2-(methylamino)acetamide asan off-white solid (67%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (brs, 1H),10.10 (s, 1H), 8.23 (s, 1H), 8.14-8.13 (m, 1H), 8.01 (s, 1H), 7.47 (s,2H), 7.38 (d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 2H), 4.61-4.54 (m, 1H),4.23 (s, 1H), 2.31 (s, 3H), 1.24 (d, J=6.0 Hz, 6H). MS (ES+) m/e 339.1(M+H)⁺.

Example 45N-(1H-indazol-5-yl)-2-(4′-methoxy-[1,1′-biphenyl]-4-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(4′-methoxy-[1,1′-biphenyl]-4-yl)-2-(methylamino)acetamideas a white solid (34%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (brs, 1H),10.14 (s, 1H), 8.21 (s, 1H), 8.14 (t, J=1.6 Hz, 1H), 8.01 (s, 1H),7.62-7.54 (m, 6H), 7.48-7.46 (m, 2H), 7.03-7.00 (m, 2H), 4.31 (s, 1H),3.79 (s, 3H), 2.34 (s, 3H). MS (ES+) m/e 387.0 (M+H)⁺.

Example 46N-(1H-indazol-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-((2-methoxyethyl)amino)-2-(3-methoxyphenyl)acetamideas an off-white solid (16%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.07 (s, 1H),10.61 (s, 1H), 9.55 (s, 2H), 8.07 (d, J=7.6 Hz, 2H), 7.52 (d, J=8.8 Hz,1H), 7.45-7.36 (m, 2H), 7.25-7.21 (m, 2H), 7.07-7.04 (m, 1H), 5.07 (s,1H), 3.80 (s, 3H), 3.78-3.59 (m, 2H), 3.31 (s, 3H), 3.11 (m, 1H), 2.99(m, 1H). MS (ES+) m/e 355.0 (M+H)⁺.

Example 472-(2-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general protocol E. The finalresidue was purified by reverse phase preparative HPLC to afford2-(2-chlorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as anoff-white solid (32%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.03 (brs, 1H),10.14 (s, 1H), 8.17-8.14 (m, 2H), 8.02 (s, 1H), 7.61-7.58 (m, 1H),7.50-7.46 (m, 3H), 7.37-7.30 (m, 2H), 4.67 (s, 1H), 2.35 (s, 3H). MS(ES+) m/e 315.0 (M+H)⁺.

Example 482-(4-fluoro-3-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford2-(4-fluoro-3-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamideas a white solid (49%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.00 (s, 1H), 10.15(s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.50-7.44 (m, 2H),7.34 (dd, J=8.4, 2.0 Hz, 1H), 7.21-7.16 (m, 1H), 7.07-7.03 (m, 1H), 4.31(s, 1H), 3.86 (s, 3H), 2.32 (s, 3H). MS (ES+) m/e 329.1 (M+H)⁺.

Example 49 2-(ethylamino)-N-(1H-indazol-5-yl)-2-(m-tolyl)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford2-(ethylamino)-N-(1H-indazol-5-yl)-2-(m-tolyl)acetamide as a yellowsolid (30%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.08 (s, 1H), 10.66 (s, 1H),9.44-9.37 (m, 2H), 8.10-8.06 (m, 2H), 7.52 (d, J=8.8 Hz, 1H), 7.47-7.36(m, 4H), 7.31 (d, J=7.6 Hz, 1H), 5.04 (t, J=6.0 Hz, 1H), 2.98-2.84 (m,2H), 2.36 (s, 3H), 1.23 (t, J=7.2 Hz, 3H). MS (ES+) m/e 309.1 (M+H)⁺.

Example 50N-(1H-indazol-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)-phenyl)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-(3-(trifluoromethoxy)phenyl)acetamideas a white solid (27%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.00 (brs, 1H),10.18 (s, 1H), 8.18 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.54-7.44 (m,5H), 7.30-7.28 (m, 1H), 4.34 (s, 1H), 2.30 (s, 3H). MS (ES+) m/e 365.0(M+H)⁺.

Example 512-(ethylamino)-2-(3-fluorophenyl)-N-(1H-indazol-5-yl)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford2-(ethylamino)-2-(3-fluorophenyl)-N-(1H-indazol-5-yl)acetamide as awhite solid (21%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.16 (s,1H), 8.18 (s, 1H), 8.11 (d, J=0.8 Hz, 1H), 8.01 (d, J=0.8 Hz, 1H),7.50-7.33 (m, 5H), 7.14-7.09 (m, 1H), 4.43 (s, 1H), 2.60-2.53 (m, 2H),1.09 (t, J=7.2 Hz, 3H). MS (ES+) m/e 313.1 (M+H)⁺.

Example 52 N-(1H-indazol-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-(m-tolyl)acetamide as a whitesolid (11%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.11 (s, 1H),8.20 (s, 2H), 8.13 (s, 1H), 8.01 (s, 1H), 7.49-7.45 (m, 2H), 7.32-7.23(m, 3H), 7.11 (d, J=7.2 Hz, 1H), 4.25 (s, 1H), 2.31 (s, 6H). MS (ES+)m/e 295.1 (M+H)⁺.

Example 532-(3-fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford2-(3-fluorophenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide as awhite solid (11%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.14 (s,1H), 8.21 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 7.50-7.32 (m, 5H),7.14-7.10 (m, 1H), 4.30 (s, 1H), 2.30 (s, 3H). MS (ES+) m/e 299.1(M+H)⁺.

Example 54(R)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford(R)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide asa white solid (17%). Enantiomers were separated by SFC (DAICEL CHTRALPAKAD column). Mobile phase: 55% EtOH with 0.1% NH₄OH in CO₂, flow rate 70g/min to afford the desired compound with 99% enantiomeric purity. ¹HNMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.12 (s, 1H),8.00 (s, 1H), 7.47 (s, 2H), 7.26 (t, J=8.0 Hz, 1H), 7.09-7.05 (m, 2H),6.85 (dd, J=8.0, 2.0 Hz, 1H), 4.21 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H).MS (ES+) m/e 311.1 (M+H). [α]²⁵C D=+98.86 (c=0.2 in MeOH).

Example 55(S)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford(S)-N-(1H-indazol-5-yl)-2-(3-methoxyphenyl)-2-(methylamino)acetamide asa white solid (12%). Enantiomers were separated by SFC (DAICEL CHTRALPAKAD column). Mobile phase: 55% EtOH with 0.1% NH₄OH in CO₂, flow rate 70g/min to afford the desired compound with 99% enantiomeric purity. ¹HNMR (400 MHz, DMSO-d₆) δ 12.97 (s, 1H), 10.05 (s, 1H), 8.12 (s, 1H),8.00 (s, 1H), 7.47 (s, 2H), 7.26 (t, J=8.0 Hz, 1H), 7.09-7.05 (m, 2H),6.85 (dd, J=8.0, 2.4 Hz, 1H), 4.20 (s, 1H), 3.75 (s, 3H), 2.30 (s, 3H).MS (ES+) m/e 311.1 (M+H)⁺. [α]^(25° C.) _(D)=−96.44 (c=0.2 in MeOH).

Example 56N-(1H-indazol-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to affordN-(1H-indazol-5-yl)-2-(methylamino)-2-(4-(trifluoromethoxy)phenyl)acetamideas a brown solid (19%). ¹H NMR (400 MHz, DMSO-d₆) δ12.99 (s, 1H), 10.15(s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.62 (d, J=8.8 Hz,2H), 7.50-7.45 (m, 2H), 7.36 (d, J=8.4 Hz, 2H), 4.30 (s, 1H), 2.30 (s,3H). MS (ES+) m/e 365.1 (M+H)⁺.

Example 572-(3-fluoro-4-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford2-(3-fluoro-4-methoxyphenyl)-N-(1H-indazol-5-yl)-2-(methylamino)acetamideas a white solid (29%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1H), 10.07(s, 1H), 8.18 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.49-7.44 (m, 2H),7.35 (dd, J=12.8, 1.2 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H), 7.16-7.12 (m,1H), 4.21 (s, 1H), 3.82 (s, 3H), 2.28 (s, 3H). MS (ES+) m/e 329.0(M+H)⁺.

Example 582-(3-chlorophenyl)-2-(ethylamino)-N-(1H-indazol-5-yl)acetamide

The reaction was conducted following general procedure F. The finalresidue was purified by reverse phase preparative HPLC to afford2-(3-chlorophenyl)-2-(ethylamino)-N-(1H-indazol-5-yl)acetamide as abrown solid (19%). ¹H NMR (400 MHz, CD₃OD) δ 8.53 (s, 1H), 8.08 (d,J=1.2 Hz, 1H), 8.02 (s, 1H), 7.62 (s, 1H), 7.53-7.39 (m, 5H), 4.55 (s,1H), 2.83-2.68 (m, 2H), 1.24 (t, J=7.2 Hz, 3H). MS (ES+) m/e 329.1(M+H)⁺.

Comparative Example 1

Comparative Example 2

Comparative Example 3

Comparative Example 4

Comparative Example 5

Comparative Example 6

Comparative Example 7

Example 59

Determination of compounds ROCK inhibitory activity in vitro (Z'lyteassay): recombinant ROCK1 (amino acids 1-535) and ROCK2 (amino acids1-552) proteins were purchased from ThermoFisher Scientific. Compoundsactivities were measured by Z′-lyte kinase kit (ThermoFisher Scientific)and IC50s were calculated.

Determination of compounds ROCK inhibitory activity in A7R5 cells: rataortic smooth muscle cell line A7R5 cells were maintained and treated inDMEM medium with 10% of fetal bovine serum. Cells were seeded withdensity of 5,000 cells/well in 96-well plates for 24 hours andsubsequently treated 90 min with testing compounds. Cells were thenfixed and processed according to the In-Cell ELISA ColorimetricDetection Kit manual (Thermo Scientific). Cellular phospho-myosin lightchains (ppMlc, Thr18/Ser19) levels were determined after treatments ofDMSO control or test compounds using the In-Cell ELISA kit. The percentinhibition rate was calculated by applying the data obtained with 1 μM(or 10 μM) compound treatment to the formula, [1-(compound/DMSO)]×100%.ppMlc data obtained with 9 points 2-fold serial dilution of compoundswere applied to the nonlinear regression curve fit function of theGraphPad Prism software to calculate the In cell IC50s.

NIH3T3 cells Acta2-promoter driven-luciferase assay: A NIH3T3 cell linestably expressing a luciferase reporter driven by the human ACTA2 genepromoter (−1000-1 bp) was established (NIH3T3-Acta2-luciferase). Thecells were plated to confluence and treated with the test compounds plusTGF 1 for 24 hr. Cells were then lysed and luciferase activity wasmeasured using the LightSwitch luciferase kit from Active Motif.

ROCK inhibitors potently inhibited ROCK kinase activity in vitro and incells. As shown in FIG. 1, ROCK inhibitors of the invention, at lowerthan 20 nanomolar concentrations, potently inhibited both isoforms ofROCK kinases activity in vitro measured by Z′-Lyte kit.

Compounds of the invention inhibited ROCKs in cells as measured by A7R5in-cell Elisa assay. A7R5 cells were treated with 9 points 2 fold serialdilution of compounds and ppMlc (T18/S19) levels were determined inorder to calculate cellular IC50s of the compounds. Results are providedin Table 1, below.

TABLE 1 ppMIc IC50 Compound (nM) Example 2  304 Example 14 207 Example15 124 Example 47 461

ROCK inhibitors of the present invention inhibit both isoforms of ROCK.A CRISPR/CAS9 system was used to create HCT116 cells containing a knockout for either ROCK1 or ROCK2. The ROCK1KO and ROCK2KO cells weretreated with the Example 2 compound for 90 min and pMypt (T853) levelswere visualized by western blotting. The ROCK inhibitor efficientlyblocked ROCK target MYPT1 phosphorylation at 110 nM. See FIG. 2.

Table 2, below, provides in vitro and cellular inhibition of ROCKs bycompounds of the invention. Compounds activities were measured byZ′-lyte kinase kit (ThermoFisher Scientific). The percent inhibitionrate was calculated by normalizing the kinase activity value obtainedwith 1 μM compound treatment against DMSO control value. IC50s werecalculated using the GraphPad Prism software with kinase activities datathat were collected from points 9 serial dilution of compoundstreatment. The ROCK inhibition in A7R5 cells and NIH3T3 (Acta2-Luc)cells was performed as described above.

Two steps of measurements were adopted for pMLC/A7R5IC50. Percentinhibition rates were calculated with a single dose (1 μM or 10 μM)compounds treatment to screen for active compounds and then cellularIC50s were measured only in compounds that have over 50% of inhibitionrates.

TABLE 2 pMLC NIH3T3, % inh Acta2- ROCK2 ROCK2 ROCK1 ROCK1 or IC₅₀ luc, %inh IC₅₀ % inh IC50 (nM) IC₅₀ Ex. # (3 μM)* (nM) (3 μM)* (nM) A7R5 (nM)24 98.8 3.6 99 3.892 224 321 29 98.8 6.3 99 65.7 934 25 98.8 9.7 99 10.6633 36 98.8 9.9 99 24.7 766 15 10 13 124 219 26 98.8 12 99 24 261 845 3098.8 13 99 16 502 960 16 99 15 93.2 3.8 252 915 @ 1 μM 42 16 8.8 454 88241 99.5 17 3.2 304 539 @ 1 μM 2 98.7 21 98.8 25 304.0 885 50 93 21 93 @0.5 μM @ 0.5 μM 28 98.8 21.2 99 17.78 834 38 100 27.4 100 61.44 1342 2395.3 43 94.3 495 1056 27 66 79 2977 48 76 71 87 @ 0.5 μM @ 0.5 μM 43 9873 4% inh. @ 1 μM @ 10 μM 37 98.8 74.3 99 98.1 2601 5 77 38 35810 49 7687 88 @ 0.5 μM @ 0.5 μM 47 135 8.3 461 659 20 94.6 152 18.4 561 2507 1996.6 160 21.4 173 1604 39 213 250 4205 17 96.3 248 93.3 <10 556 34 43849% inh. @ 10 μM 40 453 143 37% inh. @ 10 μM 44 455 42% inh. @ 10 μM 711 1628 16 2% inh. @ 1 μM @ 0.5 μM @ 10 μtM 4 >1000 >1000 18% inh. @ 10μM 31 >1000 228 35% inh. @ 10 μM 6 >1000 421 1% inh. @ 10 μM 32 >1000310 5% inh. @ 10 μM 21 95.8 91.5 2386 22 96.7 94.3 1802 18 94.1 89.62507 8 76.7 2620 @ 1 μM 33 66.5 48% inh. @ 1 μM @ 10 μM 45 22 4% inh. @1 μM @ 10 μM 46 67 27% inh. @ 1 μM @ 10 μM 14 100 32.6 207 678 @ 0.5 μM35 71 16% inh. @ 1 μM @ 1 μM 3 0 0 25% inh. @ 1 μM 13 11% inh. @ 1 μM 94% inh. @ 1 μM 10 1% inh. @ 1 μM 11 1% inh. @ 1 μM 12 1% inh. @ 1 μM 5153 1660 5453 52 34 532 1512 53 41 914 1159 54 14 200 268 55 30 589 77356 46 546 666 57 95% 25 339 473 @ 0.5 μM 58 61% 64 85% 722 1090 @ 0.5 μM@ 0.5 μM Comp1 85.7 10.6 34% inh. @ 1 μM @ 10 μM Comp2 97.5 92.2 4410Comp3 97.4 91.5 10920 Comp4 96.5 95.8 6158 @ 1 μM Comp5 96.2 33 99 1310@ 1 μM Comp6 98.8 41.76 99 41.32 6990 Comp7 100 303 100 166 18390 * %Inhibtion in ROCK1/ROCK2 assays was testing at 3 μM unless otherwisenoted.

Comparative Examples lack an alkyl substituent on the 2-amino group(i.e., the 2-amino group is NH2 in the comparative compounds), which ispresent in compounds of the invention. The addition of the 2-alkylamino(i.e., R¹ is alkyl, etc.) is associated with enhanced cellular ROCKactivity, particularly for lower alkyl amines at the 2-position (i.e.,R¹ is lower alkyl). Compare, for example, Comparative Example 2 (with aprimary 2-amino) with Examples 16, 17, and 18 that have a secondary2-alkyl amino, and particularly where the alkyl group is small. All fourcompounds show high in vitro inhibition of ROCK1 and ROCK 2, butExamples 16, 17, and 18 have enhanced cellular ROCK inhibition asmeasured by phosphorylation of the ROCK target pMLC in A7R5 cells. Forexample, Example 2 provides cellular ROCK inhibition with an IC50 over17 times lower than Comparative Example 1 (IC50s of 252 vs 4410,respectively). Similarly, the cellular IC50 for Examples 24, 25, 26, 27,and 36 are lower than Comparative Example 4, which has a primary 2-aminogroup.

Example 59

ROCK inhibitors improved oligodendrocyte process in humanoligodendrocyte/neuron progenitor cells in culture. Humanoligodendrocyte/neuron progenitor cells were cultured in vitro with orwithout ROCK inhibitor for 2 & 14 days. Nestin and MAP2 proteins werevisualized by staining with commercial antibodies to identify differentstages of neuronal cell differentiation. ROCK inhibitors of the presentinvention significantly facilitated mature neuronal cell marker MAP2expression while improved neurite outgrowth, as evidenced by strongincrease in MAP2 signal in cells differentiated in the presence of ROCKinhibitor (compound of Example 2). See FIG. 3.

Example 60

ROCK inhibitor improved neurite extension and axon ensheathment inoligodendrocytes with rat dorsal root ganglia (DRG) explants co-culturesystem. Under co-cultured condition of rat oligodendrocytes with ratdorsal root ganglia (DRG) explants, ROCK inhibitor treatment, as shownin FIG. 4, changed cytoskeletal organization, producing many shorter,ordered myelin segments, which were identified by the neurofilamentstaining. At the same time, the ROCK inhibitor also facilitated axonalsupport of oligodentrocye by organized manner, which was visualized bystaining the mature oligodendrocyte marker MBP.

Example 61

ROCK Inhibitors Penetrated Blood-Brain Barrier.

In a mouse pharmacokinetic study, 2 hours after dosing, animal tissueswere harvested to determine the compound distribution. As shown in Table3, the Example 2 Compound had excellent BBB penetrant property.

TABLE 3 Dose Plasma Brain Brain/ Compound (mg base/ ConcentrationConcentration Plasma ID Route kg) [ng/mL]* [ng/g]* Ratio Example 2 IV 10211 699 3.31 PO 10 405 700 1.73 PO 30 1400 2856 2.04 PO 100 9288 193632.08 * Plasma and brain were collected 2 hours post-dose

Brain and plasma concentrations of select ROCK inhibitors were alsoevaluated in mouse by IPLC/MS/MS at 15 min and 2 hrs following 2.5 mg/kgIV drug administration. The results are provided in Table 4, below.

TABLE 4 Mean Mean Mean Mean Conc. Conc. Brain/ Brain/ Com- at 0.25 hr at2 hr Plasma Plasma pound Dose (ng/mL) (ng/mL) Ratio Ratio ID Level RouteSite or (ng/g) or (ng/g) at 0.25 hr at 2 hr Exam- 2.5 IV Plasma 394 41.42.17 6.99 ple Brain 853 288 15 Exam- 2.5 IV Plasma 427 2.14 1.33 8.41ple Brain 566 17.7 41 Brain 541 76.6

BIBLIOGRAPHY

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1. A compound having the formula I

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; R² is selected from thegroup consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each ofwhich may be unsubstituted or optionally substituted with 1 to 3substituents independently selected from the group consisting of halo,hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl,C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,RO₂C—, aryl-O— and heteroaryl-O—; alternatively, R¹ and R² takentogether form a monocyclic group or a bicyclic group, wherein themonocyclic group has 4 to 7 ring atoms, including up to 2 ringheteroatoms, and the bicyclic group has 8 to 10 ring atoms, including upto 3 ring heteroatoms, and wherein the monocyclic group and bicyclicgroup are unsubstituted or are optionally substituted with 1 to 3substituents independently selected from the group consisting of halo,hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoroalkyl, C₁-C₃ perfluoro alkoxy, carboxyl, aryl and heteroaryl; R³ isselected from H, lower alkyl, substituted lower alkyl, and RR′N—(C₂₋₄alkyl)-; R⁴ is selected from the group consisting of H, halo, hydroxy,lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RR′N—(C₂₋₄alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—; R⁵ is selected from H, lower alkyland C₃-C₆ cycloalkyl; a is 0 or 1; b is 0 to 2; and each R and R′ isindependently selected from H, lower alkyl, and C3-C6 cycloalkyl, oralternatively, R and R′ taken together form a 5 to 6 memberedheterocyclic ring.
 2. The compound according to claim 1, having theformula II

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; R² is selected from thegroup consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each ofwhich may be unsubstituted or optionally substituted with 1 to 3substituents independently selected from the group consisting of halo,hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl,C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,RO₂C—, aryl-O— and heteroaryl-O—; alternatively, R¹ and R² takentogether form a monocyclic group or a bicyclic group, wherein themonocyclic group has 4 to 7 ring atoms, including up to 2 ringheteroatoms, and the bicyclic group has 8 to 10 ring atoms, including upto 3 ring heteroatoms, and wherein the monocyclic group and bicyclicgroup are unsubstituted or are optionally substituted with 1 to 3substituents independently selected from the group consisting of halo,hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoroalkyl, C₁-C₃ perfluoro alkoxy, carboxyl, aryl and heteroaryl; R³ isselected from H, lower alkyl, substituted lower alkyl, and RR′N—(C₂₋₄alkyl)-; R⁴ is selected from the group consisting of H, halo, hydroxy,lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RR′N—(C₂₋₄alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—; b is 0 to 2; and each R and R′ isindependently selected from H, lower alkyl, and C3-C6 cycloalkyl, oralternatively, R and R′ taken together form a 5 to 6 memberedheterocyclic ring.
 3. The compound according to claim 1, having theformula III:

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; R² is selected from thegroup consisting of aryl, heteroaryl, aralkyl, and heterocyclyl, each ofwhich may be unsubstituted or optionally substituted with 1 to 3substituents independently selected from the group consisting of halo,hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl,C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—,RO₂C—, aryl-O— and heteroaryl-O—; alternatively, R¹ and R² takentogether form a monocyclic group or a bicyclic group, wherein themonocyclic group has 4 to 7 ring atoms, including up to 2 ringheteroatoms, and the bicyclic group has 8 to 10 ring atoms, including upto 3 ring heteroatoms, and wherein the monocyclic group and bicyclicgroup are unsubstituted or are optionally substituted with 1 to 3substituents independently selected from the group consisting of halo,hydroxy, lower alkyl, lower alkoxy, amino, nitro, cyano, C₁-C₃ perfluoroalkyl, C₁-C₃ perfluoro alkoxy, carboxyl, aryl and heteroaryl; R³ isselected from H, lower alkyl, substituted lower alkyl, and RR′N—(C₂₋₄alkyl)-; and each R and R′ is independently selected from H, loweralkyl, and C₃-C₆ cycloalkyl, or alternatively, R and R′ taken togetherform a 5 to 6 membered heterocyclic ring.
 4. The compound according toclaim 1, having the formula IV:

wherein: ring A is a 5- or 6-membered aromatic ring which optionallycontains up to 3 ring heteroatoms; R³ is selected from H, lower alkyl,substituted lower alkyl, and RR′N—(C₂₋₄ alkyl)-; R⁴ is selected from thegroup consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro,cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—,RR′NCO—, RCONH—, RCONR′—, RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;b is 0 to 2; R⁶ is selected from the group consisting of H, halo, loweralkyl, substituted lower alkyl, lower alkoxy, amino, hydroxyl, andcarboxyl; R⁷ is selected from the group consisting of H, halo, hydroxy,lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, and RCONR′—; each Rand R′ is independently selected from H, lower alkyl, and C₃-C₆cycloalkyl, or alternatively, R and R′ taken together form a 5 to 6membered heterocyclic ring; and m is 1 to
 3. 5. The compound accordingto claim 1, having the formula V:

wherein: ring B is a 5- or 6-membered aromatic ring which optionallycontains up to 3 ring heteroatoms; R³ is selected from H, lower alkyl,substituted lower alkyl, and RR′N—(C₂₋₄ alkyl)-; R⁴ is selected from thegroup consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro,cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—,RR′NCO—, RCONH—, RCONR′—, RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;b is 0 to 2; R⁸ is selected from the group consisting of H, halo, loweralkyl, substituted lower alkyl, lower alkoxy, amino, hydroxyl andcarboxyl; R⁹ is selected from the group consisting of H, halo, hydroxy,lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃perfluoro alkoxy and carboxyl, RR′N—, RR′NCO—, RCONH—, and RCONR′—; eachR and R′ is independently selected from H, lower alkyl, and C₃-C₆cycloalkyl, or alternatively, R and R′ taken together form a 5 to 6membered heterocyclic ring; and m is 1 to
 3. 6. The compound accordingto claim 1, having the formula VI:

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; R³ is selected from H,lower alkyl, substituted lower alkyl, and RR′N—(C₂₋₄ alkyl)-; R⁴ isselected from the group consisting of H, halo, hydroxy, lower alkyl,lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoroalkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RR′N—(C₂₋₄ alkyl)-,and RR′N—(C₂₋₄ alkyl)-O—; b is 0 to 2; each R⁵ is independently selectedfrom the group consisting of H, halo, hydroxy, lower alkyl, loweralkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro alkoxy,carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RO₂C—, aryl-O— andheteroaryl-O—; and n is 0 to
 3. 7. The compound according to claim 1,having the formula VII:

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; R⁴ is selected from thegroup consisting of H, halo, hydroxy, lower alkyl, lower alkoxy, nitro,cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—,RR′NCO—, RCONH—, RCONR′—, RR′N—(C₂₋₄ alkyl)-, and RR′N—(C₂₋₄ alkyl)-O—;b is 0 to 2; each R⁵ is independently selected from the group consistingof H, halo, hydroxy, lower alkyl, lower alkoxy, nitro, cyano, C₁-C₃perfluoro alkyl, C₁-C₃ perfluoro alkoxy, carboxyl, RR′N—, RR′NCO—,RCONH—, RCONR′—, RO₂C—, aryl-O— and heteroaryl-O—; and n is 0 to
 3. 8.The compound according to claim 1, having the formula VIII:

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; each R⁵ is independentlyselected from the group consisting of H, halo, hydroxy, lower alkyl,lower alkoxy, nitro, cyano, C₁-C₃ perfluoro alkyl, C₁-C₃ perfluoroalkoxy, carboxyl, RR′N—, RR′NCO—, RCONH—, RCONR′—, RO₂C—, aryl-O— andheteroaryl-O—; and n is 0 to
 3. 9. The compound according to claim 1,having the formula IX:

wherein: R¹ is selected from the group consisting of lower alkyl,substituted lower alkyl, C₃-C₆ cycloalkyl, substituted C₃-C₆ cycloalkyl,R¹⁰R¹¹N(CR¹²R¹³)_(c)—, R¹⁰O(CR¹²R¹³)_(c)—, W(CR¹²R¹³)_(d)— andR¹⁰R¹¹N—C(═O)—(CR¹²R¹³)_(c)—; each R¹⁰ is independently selected from H,lower alkyl, and C₃-C₆ cycloalkyl; each R¹¹ is independently selectedfrom H, lower alkyl, and C₃-C₆ cycloalkyl; each R¹² is independentlyselected from H and lower alkyl; each R¹³ is independently selected fromH and lower alkyl; additionally or alternatively, an R¹² and an R¹³attached to the same carbon atom may be taken together to form a C₃-C₆cycloalkyl group; W is a 3- to 7-membered heterocyclic ring having 1 to3 ring heteroatoms; c is 2 to 4; d is 1 to 4; each R⁵¹ is independentlyselected from the group consisting of H, halo, hydroxy, lower alkyl,lower alkoxy, amino, C₁-C₃ perfluoro alkyl, and C₁-C₃ perfluoro alkoxy;and n is 0 to
 3. 10. A method of treating a fibrotic disorder in asubject comprising administering to the subject a therapeuticallyeffective amount of a compound according to claim
 1. 11. The method ofclaim 10, wherein fibrotic disorder is selected from the groupconsisting of pulmonary fibrosis including cystic and idiopathicpulmonary fibrosis, radiation induced lung injury, liver fibrosisincluding cirrhosis, cardiac fibrosis including arterial fibrosis,endomyocardial fibrosis, old myocardial infraction, arterial stiffness,atherosclerosis, restenosis, arthrofibrosis, Crohn's disease,myelofibrosis, Peyronie's diseases, nephrogenic systemic fibrosis,progressive massive fibrosis, retroperitoneal cavity fibrosis,schleroderma/systemic sclerosis, mediastinal fibrosis, Keloids andhypertrophic scars, glial scaring, or renal fibrosis.
 12. A method oftreating a central nervous system disorder in a subject comprisingadministering to the subject a therapeutically effective amount of acompound according to claim
 1. 13. The method of claim 12, wherein thecentral nervous system disorder is selected from the group consisting ofHuntington's disease, Parkinson's Disease, Alzheimer's, Amyotrophiclateral sclerosis (ALS), Batten disease, dementia, spinal muscularatrophy, motor neurone diseases, spinocerebellar ataxia, acute orchronic pain, dementia, neuronal degeneration, spinal cord injury,cerebral vasospasm or multiple sclerosis.
 14. A method of treatingglaucoma in a subject comprising administering to the subject atherapeutically effective amount of a compound according to claim
 1. 15.A method of treating inflammation in a subject comprising administeringto the subject a therapeutically effective amount of a compoundaccording to claim 1.